Lighting Systems and Methods of Using Lighting Systems for In Virto Potency Assay for Photofrin

ABSTRACT

Presently disclosed is a lighting system and methods of using the lighting system for in vitro potency assay for photofrin. The lighting system includes a lamp housing, a first lens, an infrared absorbing filter, an optical filter, and a second lens. The lamp housing includes a lamp and a light-port. In operation, broad spectrum light from the lamp exits the lamp housing by passing through the light-port. The first lens then collimates the broad spectrum light that exits the lamp housing through the light-port. The infrared absorbing filter then passes a first portion of the collimated broad spectrum light to the optical filter and absorbs infrared light of the broad spectrum light. The optical filter then passes a second portion of the collimated broad spectrum light to the second lens. The second lens then disperses the second portion of the collimated light to provide uniform irradiation of a cell culture plate. A method of using the lighting system for studying a photosensitizer is also disclosed.

CROSS-REFERENCE TO RELATED APPLICATION

This application claims the benefit of U.S. Provisional Application No.61/654,375 filed Jun. 1, 2012, which is entitled “In Vitro Potency Assayfor Photofrin.” This application incorporates U.S. ProvisionalApplication No. 61/654,375 herein by reference for all purposes.

BACKGROUND

Unless otherwise indicated herein, the materials in this section are notprior art to the claims and are not admitted to be prior art byinclusion in this section.

Photodynamic therapy can be used for the treatment of neoplastic andnon-neoplastic diseases. This therapy involves the administration of aphotosensitizer, such as a porphyrin, and subsequent irradiation of thecells treated with the photosensitizer at a proper dose and wavelength.

Photofrin® is an FDA-approved porphyrin-based anti-neoplastic agent thataccumulates in tumor tissue following systematic administration where itcan then be site-specifically irradiated with light at 630 nanometerscausing the formation of reactive oxygen species and the death of thesurrounding tumor mass. Before being released for clinical use,manufactured lots of Photofrin® must be shown to possess the expecteddegree of activity, i.e., the potency of the lot must be verified. Lotrelease assays for biologic drug activity, including potency assays, caneither be in vivo or in vitro assays and should measure the activity ofthe drug thought to be responsible for the clinical effect.

SUMMARY

In one respect, the present invention provides a lighting systemcomprising: a lamp housing including a lamp and a light-port, whereinbroad spectrum light from the lamp exits the lamp housing through thelight-port; a first lens to collimate the broad spectrum light thatexits the lamp housing through the light-port; an infrared absorbingfilter to pass a first portion of the collimated broad spectrum lightand absorb infrared light of the broad spectrum light that passesthrough the light-port and that is collimated by the first lens, whereinthe first portion of the collimated broad spectrum light comprises asecond portion of the collimated broad spectrum light; an optical filterto pass the second portion of the collimated broad spectrum light afterthe first portion of the collimated broad spectrum light reaches theoptical filter; and a second lens to disperse the second portion of thecollimated light that passed through the optical filter.

In another respect, the present invention provides a lighting system,wherein the first lens comprises a condenser lens.

In another respect, the present invention provides a lighting system,wherein the first lens comprises a Fresnel lens.

In another respect, the present invention provides a lighting system,wherein the lamp comprises a xenon arc lamp.

In another respect, the present invention provides the lighting system,wherein the optical filter comprises an infrared blocking filter and ashort pass filter.

In another respect, the present invention provides a lighting system,wherein the infrared blocking filter absorbs residual infrared light ofthe first portion of the collimated broad spectrum light, and whereinthe short pass filter filters out light of the first portion of thecollimated broad spectrum light of wavelengths greater than 650 nm.

In another respect, the present invention provides a lighting systemcomprising: a reflector to reflect the first portion of the collimatedbroad spectrum light that passes through the infrared absorbing filter.

In another respect, the present invention provides a lighting system,wherein the first portion of the collimated broad spectrum lightreflected by the reflector propagates to the infrared blocking filter,and wherein the second portion of the collimated broad spectrum lightthat propagates to the infrared blocking filter, as part of the firstportion of the collimated broad spectrum light, passes through theinfrared blocking filter and then through the short pass filter.

In another respect, the present invention provides a lighting system,wherein the first portion of the collimated broad spectrum lightreflected by the reflector propagates to the short pass filter, andwherein the second portion of the collimated broad spectrum light thatpropagates to the short pass filter, as part of the first portion of thecollimated broad spectrum light, passes through the short pass filterand then through the infrared blocking filter.

In another respect, the present invention provides a lighting system,wherein the reflector comprises a dichroic mirror.

In another respect, the present invention provides a lighting system,wherein the dichroic mirror absorbs at least a portion of infrared lightthat passes through the light-port and the infrared absorbing filter.

In another respect, the present invention provides a lighting system,wherein the optical filter comprises a band-pass filter.

In another respect, the present invention provides a lighting system,wherein the lamp housing is sealed so that the broad spectrum light fromthe lamp exits the lamp housing only through the light-port.

In another respect, the present invention provides a lighting system,wherein the lamp housing is sealed so that less than 1% of the broadspectrum light from the lamp exits the lamp housing other than throughthe light-port.

In another respect, the present invention provides a lighting systemcomprising: a ring stand; a first support ring removably attached to thering stand; and a base.

In another respect, the present invention provides a lighting system,wherein the second lens comprises a first dispersing lens and a seconddispersing lens; and wherein the first support ring holds the firstdispersing lens and the second dispersing lens in place.

In another respect, the present invention provides a lighting system,wherein the first dispersing lens comprises a first plano convex lens;wherein the second dispersing lens comprises a second plano convex lens;wherein the first plano convex lens comprises a first plano side and afirst convex side; wherein the second plano convex lens comprises asecond plano side and a second convex side; and wherein the first convexside is adjacent to the second convex side with a gap between the firstconvex side and the second convex side.

In another respect, the present invention provides a lighting system,wherein the gap is within the range of 2 millimeters and 4 millimeters,inclusive.

In another respect, the present invention provides a lighting system,wherein the gap is 3 millimeters.

In another respect, the present invention provides a lighting system,wherein the infrared absorbing filter comprises an infrared absorbingliquid filter.

In another respect, the present invention provides a lighting system,wherein the infrared absorbing liquid filter absorbs between 90% and100%, inclusive, of the infrared light of the broad spectrum light thatthat passes through the light-port.

In another respect, the present invention provides a lighting systemcomprising: a wall, wherein the light-port is located within the wall,and wherein a position of the second lens is adjustable in at least oneof a direction parallel to the wall and a direction perpendicular to thewall.

In another respect, the present invention provides a lighting systemcomprising: a shelf, and a lens slider including a first hole forpassing light; wherein the shelf comprises a shelf riser parallel to thewall, wherein the shelf comprises a shelf top perpendicular to the wall,wherein the shelf top includes a second hole for passing light, whereinthe second lens is removably attached to the lens slider, wherein thelens slider is removably attached to the shelf top, and wherein at leasta portion of the first hole is above or below at least a portion of thesecond hole.

In another respect, the present invention provides a lighting system,wherein the shelf riser comprises a first parallel adjustment slot; andwherein the shelf top comprises a first perpendicular adjustment slot.

In another respect, the present invention provides a lighting system,wherein the wall comprises a base wall and a wall of the lamp housing.

In another respect, the present invention provides a lighting system,wherein the lamp housing comprises a top, wherein the light-port iswithin the top, wherein the first lens is within the light-port, andwherein the infrared absorbing filter, the optical filter, and thesecond lens are located at positions above the top.

In another respect, the present invention provides a lighting system,wherein the lamp housing comprises a bottom, wherein the light-port iswithin the bottom, wherein the first lens is within the light-port, andwherein the infrared absorbing filter, the optical filter, and thesecond lens are located at positions below the bottom.

In one aspect, the present invention provides a method comprising usinga lighting system to irradiate contents of a cell culture plate.

In another aspect, the present invention provides a method of using alighting system to irradiate contents of a cell culture plate, whereinthe cell culture plate comprises a photosensitizer.

In another aspect, the present invention provides a method for studyinga photosensitizer comprising: adding the photosensitizer to a portion ofwells on a cell culture plate to form photosensitizer assay wells, thewells comprising carcinoma cells; incubating the photosensitizer assaywells for a first predetermined time period; optionally washing thephotosensitizer assay wells; irradiating the photosensitizer assay wellswith a lighting system at a predetermined wavelength to form irradiatedwells, wherein each well is uniformly irradiated; incubating theirradiated wells for a second predetermined time period; and determiningpercent viability of the carcinoma cells contained in the wells.

In yet another aspect, the present invention provides a method forstudying a photosensitizer, wherein the step of washing thephotosensitizer assay wells is mandatory.

In another aspect, the present invention provides a method for studyinga photosensitizer, wherein a remaining portion of the wells are controlwells.

In another aspect, the present invention provides a method for studyinga photosensitizer, wherein a portion of the wells contain a referencedrug used for comparison.

In another aspect, the present invention provides a method for studyinga photosensitizer, wherein the photosensitizer is a porphyrin-basedanti-neoplastic agent.

In another aspect, the present invention provides a method for studyinga photosensitizer, wherein the porphyrin-based anti-neoplastic agent isporfimer sodium.

In yet another aspect, the present invention provides a method forstudying a photosensitizer, wherein the photosensitizer assay wells areirradiated with light within a range of 400 nm to 650 nm.

In another aspect, the present invention provides a method for studyinga photosensitizer, wherein the carcinoma cells are A549 human lungcarcinoma cells.

In another aspect, the present invention provides a method for studyinga photosensitizer, wherein the irradiating step is standardized.

In another aspect, the present invention provides a method for studyinga photosensitizer, wherein the cell culture plate is an opaque blackplate.

These as well as other aspects, advantages, and alternatives will becomeapparent to those of ordinary skill in the art by reading the followingdetailed description, with reference where appropriate to theaccompanying drawings. Further, it should be understood that thissummary and the other description provided throughout this document isprovided to explain the invention by way of example and is not intendedto be limiting.

BRIEF DESCRIPTION OF THE DRAWINGS

Example embodiments of the invention are described herein with referenceto the drawings, wherein like parts are designated by like referencenumerals, and wherein:

FIG. 1 is a block diagram of a lighting system;

FIG. 2 is a block diagram of an alternative embodiment lighting system;

FIG. 3 is a block diagram of another embodiment lighting system;

FIG. 4 is a block diagram of another embodiment lighting system;

FIG. 5 is a perspective view of another embodiment lighting system;

FIG. 6 is a perspective view of the lens slider and related componentsof the lighting system illustrated in FIG. 5;

FIG. 7 is a perspective view of the shelf and related components of thelighting system illustrated in FIG. 5;

FIG. 8A is a side elevational view of another embodiment lightingsystem;

FIG. 8B is a front elevational view of the shelf and related componentsof the lighting system illustrated in FIG. 8A;

FIG. 8C is a perspective view of the lens slider and related componentsof the lighting system illustrated in FIG. 8A;

FIG. 9 is a front elevational view of the lighting system illustrated inFIG. 8A;

FIG. 10 is a flow chart of a method of using a lighting systemconstructed in accordance with principles disclosed herein to irradiatecontents of a cell culture plate;

FIG. 11 is a diagram of plate layout for Photofrin® potency assay;

FIG. 12 is a graphical representation of Photofrin® dose-response ofA549 cells;

FIG. 13 is a graphical representation of homogeneity of irradiance;

FIG. 14A is a graphical representation of dose response of Photofrin®from 250-0.977 μg/mL in a black plate; and

FIG. 14B is a graphical representation of dose response of Photofrin®from 250-0.977 μg/mL in a clear plate.

DETAILED DESCRIPTION OF THE INVENTION 1. Example Architecture

In this description, the articles “a” or “an” are used to introduceelements of the example embodiments. The intent of using those articlesis that there is one or more of the elements. The intent of using theconjunction “or” within a described list of at least two terms is toindicate any of the listed terms or any combination of the listed terms.The use of ordinal numbers such as “first,” “second,” “third” and so onis to distinguish respective elements rather than to denote a particularorder of those elements.

FIG. 1 illustrates an embodiment of a lighting system generallydesignated 100 and components thereof. The lighting system 100 includesa lamp housing 102, a first lens 108, an infrared absorbing filter 110,a reflector 112, an optical filter 114, a second lens 116, and a cellculture plate 120. The lamp housing 102 includes a lamp 104 and alight-port 106.

In the lighting system 100, the first lens 108 is within the light-port106. The infrared absorbing filter 110 is connected to the light-port106. The reflector 112 is connected to the infrared absorbing filter110. The optical filter 114 is connected to the reflector 112. Thesecond lens 116 is located a distance 118 from the optical filter 114.The cell culture plate 120 is located a distance 122 from the opticalfilter 114.

In operation, the cell culture plate 120, as well as contents of thecell culture plate 120, is irradiated with light from the lamp 104. Thelamp 104 may be one or more 1000 watt xenon arc lamps. First, broadspectrum light from the lamp 104 exits the lamp housing 102 by passingthrough the light-port 106. The lamp housing 102 may be sealed so thatthe broad spectrum light from the lamp 104 exits the lamp housing 102only through the light-port 106. Alternatively, the lamp housing 102 maybe sealed so that less than 1% of the broad spectrum light from the lamp104 exits the lamp housing 102 other than through the light-port 106.

After the broad spectrum light exits the lamp housing 102 by passingthrough the light-port 106, the broad spectrum light is collimated bythe first lens 108. The first lens 108 can be arranged in variousconfigurations. In a first configuration, the first lens 108 may be a 50millimeter (mm) focal length condenser lens. In that first configurationor in another configuration, the first lens 108 may be a Fresnel lens.Moreover, in that first configuration or in another configuration, thefirst lens 108 may have a diameter of 2 or 3 inches (in). Other examplesof the diameter of the first lens 108 are possible. In a second exampleconfiguration, the first lens 108 can comprise two or more lenses. Inaccordance with one or more of these example embodiments of the firstlens 108, the first lens 108 can comprise a lens holder configured forsupporting a lens, such as a glass lens, and for connecting the firstlens 108 to another component of the lighting system 100, such asinfrared absorbing filter 110, the light-port 106, the reflector 112, ora spacer.

After the broad spectrum light is collimated by the first lens 108, thebroad spectrum light reaches the infrared absorbing filter 110. Theinfrared absorbing filter 110 absorbs infrared light of the broadspectrum light. The infrared absorbing filter 110 also passes a firstportion of the collimated broad spectrum light to the reflector 112.

After the first portion of the collimated broad spectrum light is passedby the infrared absorbing filter 110 to the reflector 112, the reflector112 reflects the first portion of the collimated broad spectrum light.The first portion of the collimated broad spectrum light then propagatesto the optical filter 114. Alternatively, the reflector 112 may reflectonly a portion of the first portion of the collimated broad spectrumlight, and the portion of the first portion of the collimated broadspectrum light then propagates to the optical filter 114. The reflector112 may be one or more dichroic mirrors. The first portion of thecollimated broad spectrum light that is reflected by the reflector 112to the optical filter 114 may have a wavelength in a given range. Forexample, the collimated broad spectrum light may have a given range of400 nanometers (nm) to 700 nm. Other given ranges are possible. In someembodiments, the reflector 112 absorbs at least a portion of infraredlight or at least a portion of ultraviolet light of the collimated broadspectrum light. With this arrangement, the reflector 112 functions likean optical filter by passing only visible light to the optical filter114.

After the first portion of the collimated broad spectrum lightpropagates to the optical filter 114, the optical filter 114 passes asecond portion of the collimated broad spectrum light to the second lens116. The optical filter 114 may be one or more band-pass filters. As anexample, the light passed to the second lens 116 may be collimated lightwithin the range of 400 nm to 630 nm (i.e., no infrared or ultravioletlight). Different ranges can be passed to the second lens 116 dependingon what light is reflected by the reflector 112 and passed by theoptical filter 114. As another example, the range of wavelengths can beany variety of ranges, such as between about 595 nm and about 645 nm,between about 620 nm and about 640 nm, between about 615 nm and about645 nm, or between about 610 nm and about 650 nm. Narrower band-passfilters can be used to reduce the range. The range of wavelengths can becentered on a given wavelength, such as 630 nm. A different centerwavelength can be selected by changing components of the lighting system100, such as the reflector 112 and the optical filter 114.

After the second portion of the collimated broad spectrum light ispassed by the optical filter 114 to the second lens 116, the second lens116 disperses the second portion of the collimated broad spectrum lightto the cell culture plate 120. The second lens 116 can be arranged invarious configurations. In a first configuration, the second lens 116may have a diameter of 3 in. Other examples of the diameter of thesecond lens 116 are possible. In a second example configuration, thesecond lens 116 can comprise two or more lenses. In accordance with oneor more of these example embodiments of the second lens 116, the secondlens 116 can comprise a lens holder configured for supporting a lens,such as a glass lens.

The cell culture plate 120 can be arranged in various configurations. Ingeneral, the cell culture plate 120 can have a bottom area, a top area,and a vertical wall extending from the bottom area to the top area. Thebottom area, the top area, and the vertical wall can each be transparentor opaque. For purposes of this description, transparent can be cleartransparent but is not so limited. For purposes of this description,opaque can be black opaque but is not so limited. The bottom area andvertical wall can be made of the same material such as polystyrene orsome other material. The bottom area and vertical wall can have anexternal surface and an internal surface. Contents placed within thecell culture plate can contact the internal surfaces of the bottom areaand the vertical wall.

The top area can be open or opened to allow contents to be placed withincell culture plate 120 and to allow those contents to be subsequentlyremoved. The top area can include a removable cover, such as atransparent cover. The cell culture plate 120 can comprise multiplewells, such as 2, 4, 6, 12, 24, 48, or 96 wells. The multiple wells canbe transparent or opaque. The multiple wells can be made of polystyreneor some other material. The cell culture plate 120 can be a petri dish.

The bottom area, the top area, and vertical wall can be arranged toprovide the cell culture plate 120 with a defined shape. In one respect,cell culture plate 120 can have a rectangular shape. In that regard, thevertical wall can comprise four vertical walls that define an outerboundary of the rectangular shape. In accordance with an exampleembodiment, two of the vertical walls can have a length of 12.8centimeters (cm) and a height of 1.42 cm, and the other two verticalwalls can have a length of 8.55 cm and a height of 1.42 cm. In anotherrespect, cell culture plate 120 can have a circular shape. Otherexamples of the defined shape cell culture plate 120 can take or otherexamples of vertical wall dimensions are also possible.

The distance 118 between the optical filter 114 and the second lens 116and the distance 122 between the optical filter 114 and the cell cultureplate 120 may be selected to uniformly irradiate the cell culture plate120. “Uniformly irradiate” and “uniform irradiation” refers to providinglight dispersed by the second lens 116 to the entire area of the cellculture plate 120 or to the entire bottom area of the cell culture plate120.

The distance 118 between the optical filter 114 and the second lens 116to uniformly irradiate the cell culture plate 120 may be 13.6 cm. Otherdistances between the optical filter 114 and the second lens 116 touniformly irradiate the cell culture plate 120 are possible. Thedistance 118 can be a distance between portions of optical filter 114and second lens 116 that are nearest each other. With respect to theorientation of lighting system 100 in FIG. 1, those portions can includea lower side of optical filter 114 and an upper side of second lens 116.Alternatively, the distance 118 between the optical filter 114 and thesecond lens 116 may be a distance between a vertical center point of theoptical filter 114 and a vertical center point of the second lens 116.Other examples of specifying the distance 118 between the optical filter114 and the second lens 116 are possible.

The distance 122 between the optical filter 114 and the cell cultureplate 120 to uniformly irradiate the cell culture plate 120 can bespecified in various ways. In one respect, the distance 122 can bespecified as a distance from a highest point of optical filter 114 tothe external surface of the bottom area of the cell culture plate 120.In that regard, for a first case, the distance 122 can be 95.5 cm. Inanother respect, the distance 122 can be specified as a distance fromthe highest point of optical filter 114 to the top area of the cellculture plate 120. In accordance with the first case referred to above,if the vertical wall (the distance between the top area of cell cultureplate 120 and the external surface of the bottom area of the cellculture plate 120) has a height of 1.42 cm, then the distance can be94.08 cm. In another respect, the distance 122 between the opticalfilter 114 and the cell culture plate 120 may be specified as a distancebetween a closest point of the optical filter 114 and a closest point ofthe cell culture plate 120. In yet another respect, the distance 122between the optical filter 114 and the cell culture plate 120 may bespecified as a distance between a vertical center point of the opticalfilter 114 and a vertical center point of the cell culture plate 120.Other examples of specifying the distance 122 between the optical filter114 and the cell culture plate 120 are possible.

The components connected together in the lighting system 100, or inother lighting systems described herein, can be removably connected suchthat the components can be disconnected from one another and removedfrom the lighting system for adjustment, cleaning, repair, replacementor otherwise. The portions of components that connect to one another caninclude one or more seals to prevent, or at least reduce, any light fromexiting the lighting system at those connected portions. A personskilled in the art will understand that the connection of two or morecomponents can be facilitated by use of one or more spacers to achievedesired distances between components of the lighting system. One or morespacers may be placed between components of the lighting system so asnot to impact uniform irradiation of the cell culture plate. For clarityof the figures, spacers are typically not shown.

In an alternative embodiment, the light-port 106 is connected to theinfrared absorbing filter 110. With this arrangement, the infraredabsorbing filter 110 is connected to the first lens 108, and the firstlens 108 is connected to the reflector 112.

In another embodiment, the first lens 108 is outside of the light-port106. With this arrangement, the light-port 106 may be connected to thefirst lens 108, the first lens 108 may be connected to the infraredabsorbing filter 110, and the infrared absorbing filter 110 may beconnected to the reflector 112. Alternatively, the light-port 106 may beconnected to the infrared absorbing filter 110, the infrared absorbingfilter 110 may be connected to the first lens 108, and the first lens108 may be connected to the reflector 112.

In another embodiment, the lamp housing 102, the first lens 108, theinfrared absorbing filter 110, the reflector 112, the optical filter114, and the second lens 116 are each positioned on a ledge (not shown)above a table (not shown). With this arrangement, the cell culture plate120 is then positioned on a temperature controlling device (not shown)located on the table. The temperature controlling device can be arrangedin various configurations. For instance, the temperature controllingdevice may be a heating device or a cooling device. The temperaturecontrolling device may maintain the cell culture plate 120, as well asthe contents of the cell culture plate 120, at a regulated temperature.

In yet another embodiment, the reflector 112 is inverted. With thisarrangement, the reflector 112 reflects the first portion of thecollimated broad spectrum light upward. As such, the optical filter 114,the second lens 116, and the cell culture plate 120 are each locatedabove the reflector 112. In accordance with this embodiment, uniformirradiation of the cell culture plate 120 comprises providing lightdispersed by the second lens 116 to the entire bottom area of the cellculture plate 120.

FIG. 2 illustrates an alternative embodiment lighting system generallydesignated 200 and components thereof. The lighting system 200 includesa lamp housing 202, a first lens 208, an infrared absorbing filter 210,a reflector 212, an optical filter 214, a second lens 216, a cellculture plate 220, a ring stand 224, a first support ring 226, and abase 228. The lamp housing 202 includes a lamp 204, a mirror 205, and alight-port 206.

In the lighting system 200, the first lens 208 is within the light-port206. The infrared absorbing filter 210 is connected to the light-port206. The reflector 212 is connected to the infrared absorbing filter210. The optical filter 214 is connected to the reflector 212. Thesecond lens 216 is supported by the first support ring 226. Support forthe second lens 216 by the first support ring 226 may include the secondlens 216 being on the first support ring 226. The first support ring 226is removably attached to the ring stand 224. The second lens 216 islocated a distance 218 from the optical filter 214. The first supportring 226 may be slid up the ring stand 224 to adjust the distance 218between the optical filter 214 and the second lens 216. The firstsupport ring 226 may be slid down the ring stand 224 to adjust thedistance 218 between the optical filter 214 and the second lens 216. Thebase 228 is connected to the ring stand 224. The cell culture plate 220is located a distance 222 from the optical filter 214.

Components of the lighting system 200 can be arranged like components ofthe lighting system 100 illustrated in FIG. 1. For example, the lamphousing 202 can be arranged like the lamp housing 102, the lamp 204 canbe arranged like the lamp 104, the light-port 206 can be arranged likethe light-port 106, the first lens 208 can be arranged like the firstlens 108, the infrared absorbing filter 210 can be arranged like theinfrared absorbing filter 110, the reflector 212 can be arranged likethe reflector 112, the optical filter 214 can be arranged like theoptical filter 114, the second lens 216 can be arranged like the secondlens 116, and the cell culture plate 220 can be arranged like the cellculture plate 120.

In operation, the cell culture plate 220, as well as the contents of thecell culture plate 220, is irradiated with light from the lamp 204.First, broad spectrum light from the lamp 204 exits the lamp housing 202by passing through the light-port 206. The broad spectrum light may bereflected by the mirror 205 to the light-port 206. Something other thana mirror could be used to reflect the broad spectrum light to thelight-port 206. For simplicity of describing the lighting system 200,and other lighting systems described herein, the component that reflectsthe broad spectrum light to the light-port 206 can be referred to as amirror. The mirror 205 may be one or more concave mirrors. With thisarrangement, the mirror 205 may increase the power of the broad spectrumlight passing through the light-port 206. Additionally, the mirror 205may have two or more adjustments (not shown) to allow the mirror 205 tomove in perpendicular planes. The two or more adjustments can be used tooptimize the broad spectrum light for irradiating the cell culture plate220. For instance, the two or more adjustments can be used to center thebroad spectrum light through the first lens 208. As another example, thetwo or more adjustments can be used to avoid passing a most intenseportion of the broad spectrum light through an arc of one or moreelectrodes associated with the lamp 204.

After the broad spectrum light exits the lamp housing 202 by passingthrough the light-port 206, the broad spectrum light is collimated bythe first lens 208.

After the broad spectrum light is collimated by the first lens 208, thecollimated broad spectrum light reaches the infrared absorbing filter210. The infrared absorbing filter 210 absorbs infrared light of thecollimated broad spectrum light. The infrared absorbing filter 210 alsopasses a first portion of the collimated broad spectrum light to thereflector 212.

After the first portion of the collimated broad spectrum light is passedby the infrared absorbing filter 210 to the reflector 212, the reflector212 reflects the first portion of the collimated broad spectrum light.The first portion of the collimated broad spectrum light then propagatesto the optical filter 214. Alternatively, the reflector 212 may reflectonly a portion of the first portion of the collimated broad spectrumlight, and the portion of the first portion of the collimated broadspectrum light then propagates to the optical filter 214. The reflector212 reflects light with the same given range of wavelengths as thereflector 110.

After the first portion of the collimated broad spectrum lightpropagates to the optical filter 214, the optical filter 214 passes asecond portion of the collimated broad spectrum light to the second lens216. The optical filter 214 passes light with the same given range ofwavelengths as the optical filter 114.

After the second portion of the collimated broad spectrum light ispassed by the optical filter 214 to the second lens 216, the second lens216 disperses the second portion of the collimated broad spectrum lightto the cell culture plate 220.

The distance 218 between the optical filter 214 and the second lens 216and the distance 222 between the optical filter 214 and the cell cultureplate 220 may be selected to uniformly irradiate the cell culture plate220. “Uniformly irradiate” and “uniform irradiation” refers to providinglight dispersed by the second lens 216 to the entire area of the cellculture plate 220 or to the entire bottom area of the cell culture plate220.

The distance 218 between the optical filter 214 and the second lens 216to uniformly irradiate the cell culture plate 220 may be 13.6 cm. Otherdistances between the optical filter 214 and the second lens 216 touniformly irradiate the cell culture plate 220 are possible. Thedistance 218 can be a distance between portions of optical filter 214and second lens 216 that are nearest each other. With respect to theorientation of lighting system 200 in FIG. 2, those portions can includea lower side of optical filter 214 and an upper side of second lens 216.Alternatively, the distance 218 between the optical filter 214 and thesecond lens 216 may be a distance between a vertical center point of theoptical filter 214 and a vertical center point of the second lens 216.Other examples of specifying the distance 218 between the optical filter214 and the second lens 216 are possible.

The distance 222 between the optical filter 214 and the cell cultureplate 220 to uniformly irradiate the cell culture plate 220 can bespecified in various ways. In one respect, the distance 222 can bespecified as a distance from a highest point of optical filter 214 tothe external surface of the bottom area of the cell culture plate 220.In that regard, for a first case, the distance 222 can be 95.5 cm. Inanother respect, the distance 222 can be specified as a distance fromthe highest point of optical filter 214 to the top area of the cellculture plate 220. In accordance with the first case referred to above,if the vertical wall (the distance between the top area of cell cultureplate 220 and the external surface of the bottom area of the cellculture plate 220) has a height of 1.42 cm, then the distance can be94.08 cm. In another respect, the distance 222 between the opticalfilter 214 and the cell culture plate 220 may be specified as a distancebetween a closest point of the optical filter 214 and a closest point ofthe cell culture plate 220. In yet another respect, the distance 222between the optical filter 214 and the cell culture plate 220 may bespecified as a distance between a vertical center point of the opticalfilter 214 and a vertical center point of the cell culture plate 220.Other examples of specifying the distance 222 between the optical filter214 and the cell culture plate 220 are possible.

In an alternative embodiment, the light-port 206 is connected to theinfrared absorbing filter 210. With this arrangement, the infraredabsorbing filter 210 is connected to the first lens 208, and the firstlens 208 is connected to the reflector 212.

In another embodiment, the first lens 208 is outside of the light-port206. With this arrangement, the light-port 206 may be connected to thefirst lens 208, the first lens 208 may be connected to the infraredabsorbing filter 210, and the infrared absorbing filter 210 may beconnected to the reflector 212. Alternatively, the light-port 206 may beconnected to the infrared absorbing filter 210, the infrared absorbingfilter 210 may be connected to the first lens 208, and the first lens208 may be connected to the reflector 212.

In yet another embodiment, the lamp housing 202, the first lens 208, theinfrared absorbing filter 210, the reflector 212, the optical filter214, and the second lens 216 are each positioned on a ledge (not shown)above a table (not shown). With this arrangement, the cell culture plate220 may then be positioned on a temperature controlling device (notshown) located on the table. The temperature controlling device can bearranged in various configurations. For instance, the temperaturecontrolling device may be a heating device or a cooling device. Thetemperature controlling device may maintain the cell culture plate 220,as well as the contents of the cell culture plate 220, at a regulatedtemperature.

Although FIG. 2 illustrates the cell culture plate 220 on one side ofbase 228, in an alternative arrangement, the base 228 may be rotatedabout a vertical axis of ring stand 224 such that base 228 is positionedwhere the cell culture plate 220 is shown in FIG. 2 and cell cultureplate 220 can be placed upon base 228. In yet another alternativearrangement, the ring stand 224 may be positioned in a center of thebase 228 and cell culture plate 220 is placed upon a portion of the base228 below the first support ring 226.

FIG. 3 illustrates another embodiment of a lighting system generallydesignated 300 and components thereof. The lighting system 300 includesa lamp housing 302, a first lens 312, an infrared absorbing filter 314,an optical filter 316, a second lens 318, a cell culture plate 322, aring stand 326, a base 328, and a first support ring 330. The lamphousing 302 includes a lamp 304, a light-port 306, a top 308, and abottom 310.

In the lighting system 300, the lamp 304 is within the lamp housing 302.The light-port 306 is within the bottom 310. The infrared absorbingfilter 314, the optical filter 316, and the second lens 318 are locatedat positions below the bottom 310. The first lens 312 is within thelight-port 306. The infrared absorbing filter 314 is connected to thelight-port 306. The optical filter 316 is connected to the infraredabsorbing filter 314. The second lens 318 is supported by the firstsupport ring 330. Support for the second lens 318 by the first supportring 330 may include the second lens 318 being on the first support ring330. The first support ring 330 is removably attached to the ring stand326. The second lens is located a distance 320 from the optical filter316. The first support ring 330 may be slid up the ring stand 326 toadjust the distance 320 between the optical filter 316 and the secondlens 312. The first support ring 330 may be slid down the ring stand 326to adjust the distance 320 between the optical filter 316 and the secondlens 318. The base 328 is connected to the ring stand 326. The cellculture plate 322 is located a distance 324 from the optical filter 316.

Components of the lighting system 300 can be arranged like components ofthe lighting system 100 illustrated in FIG. 1. For example, the lamphousing 302 can be arranged like the lamp housing 102, the lamp 304 canbe arranged like the lamp 104, the light-port 306 can be arranged likethe light-port 106, the first lens 312 can be arranged like the firstlens 108, the infrared absorbing filter 314 can be arranged like theinfrared absorbing filter 110, the optical filter 314 can be arrangedlike the optical filter 114, the second lens 318 can be arranged likethe second lens 116, and the cell culture plate 322 can be arranged likethe cell culture plate 120.

In operation, the cell culture plate 322, as well as the contents of thecell culture plate 322, is irradiated with light from the lamp 304.First, broad spectrum light from the lamp 304 exits the lamp housing 302by passing through the light-port 306.

After the broad spectrum light exits the lamp housing 302 by passingthrough the light-port 306, the broad spectrum light is collimated bythe first lens 312.

After the broad spectrum light is collimated by the first lens 312, thecollimated broad spectrum light reaches the infrared absorbing filter314. The infrared absorbing filter 314 absorbs infrared light of thecollimated broad spectrum light. The infrared absorbing filter 314 alsopasses a first portion of the collimated broad spectrum light to theoptical filter 316.

After the first portion of the collimated broad spectrum light is passedby the infrared absorbing filter 314 to the optical filter 316, theoptical filter 316 passes a second portion of the collimated broadspectrum light to the second lens 318. As an example, the light passedto the second lens 318 may be collimated light within the range of 400nm to 630 nm (i.e., no infrared or ultraviolet light). Narrowerband-pass filters can be used to reduce the range.

After the second portion of the collimated broad spectrum light ispassed by the optical filter 316 to the second lens 318, the second lens318 disperses the second portion of the collimated broad spectrum lightto the cell culture plate 322.

The distance 320 between the optical filter 316 and the second lens 318and the distance 324 between the optical filter 316 and the cell cultureplate 322 may be selected to uniformly irradiate the cell culture plate322. “Uniformly irradiate” and “uniform irradiation” refers to providinglight dispersed by the second lens 318 to the entire area of the cellculture plate 322 or to the entire bottom area of the cell culture plate322.

The distance 320 between the optical filter 316 and the second lens 318to uniformly irradiate the cell culture plate 322 may be 13.6 cm. Otherdistances between the optical filter 316 and the second lens 318 touniformly irradiate the cell culture plate 322 are possible. Thedistance 320 can be a distance between portions of optical filter 316and second lens 318 that are nearest each other. With respect to theorientation of lighting system 300 in FIG. 3, those portions can includea lower side of optical filter 316 and an upper side of second lens 318.Alternatively, the distance 320 between the optical filter 316 and thesecond lens 318 may be a distance between a vertical center point of theoptical filter 316 and a vertical center point of the second lens 318.Other examples of specifying the distance 320 between the optical filter316 and the second lens 318 are possible.

The distance 324 between the optical filter 316 and the cell cultureplate 322 to uniformly irradiate the cell culture plate 322 can bespecified in various ways. In one respect, the distance 324 can bespecified as a distance from a highest point of optical filter 316 tothe external surface of the bottom area of the cell culture plate 322.In that regard, for a first case, the distance 324 can be 95.5 cm. Inanother respect, the distance 324 can be specified as a distance fromthe highest point of optical filter 316 to the top area of the cellculture plate 322. In accordance with the first case referred to above,if the vertical wall (the distance between the top area of cell cultureplate 322 and the external surface of the bottom area of the cellculture plate) has a height of 1.42 cm, then the distance can be 94.08cm. In another respect, the distance 324 between the optical filter 316and the cell culture plate 322 may be specified as a distance between aclosest point of the optical filter 316 and a closest point of the cellculture plate 322. In yet another respect, the distance 324 between theoptical filter 316 and the cell culture plate 322 may be specified as adistance between a vertical center point of the optical filter 316 and avertical center point of the cell culture plate 322. Other examples ofspecifying the distance 324 between the optical filter 316 and the cellculture plate 322 are possible.

In an alternative embodiment, the first lens 312 is outside of thelight-port 306. With this arrangement, the light-port 306 may beconnected to the first lens 312, and the first lens 312 may be connectedto the infrared absorbing filter 314. Alternatively, the light-port 306may be connected to the infrared absorbing filter 314, and the infraredabsorbing filter 314 may be connected to the first lens 312.

In another embodiment, the light-port 306 may be connected to theinfrared absorbing filter 314. With this arrangement, the infraredabsorbing filter 314 is connected to the first lens 312, and the firstlens 312 is connected to the optical filter 316.

In yet another embodiment, the lamp housing 302, the first lens 312, theinfrared absorbing filter 314, the optical filter 316, and the secondlens 318 are each positioned on a ledge (not shown) above a table (notshown). With this arrangement, the cell culture plate 322 is then bepositioned on a temperature controlling device (not shown) located onthe table. The temperature controlling device can be arranged in variousconfigurations. For instance, the temperature controlling device may bea heating device or a cooling device. The temperature controlling devicemay maintain the cell culture plate 322, as well as the contents of thecell culture plate 322, at a regulated temperature.

Although FIG. 3 illustrates the cell culture plate 322 on one side ofbase 328, in an alternative arrangement, the base 328 may be rotatedabout a vertical axis of ring stand 326 such that base 328 is positionedwhere the cell culture plate 322 is shown in FIG. 3 and cell cultureplate 322 can be placed upon base 328. In yet another alternativearrangement, the ring stand 326 may be positioned in a center of thebase 328 and cell culture plate 322 is placed upon a portion of the base328 below the first support ring 330.

FIG. 4 illustrates another embodiment of a lighting system generallydesignated 400 and components thereof. The lighting system 400 includesa lamp housing 402, a first lens 412, an infrared absorbing filter 414,an optical filter 416, a second lens 418, a cell culture plate 422, aring stand 426, a base 428, a first support ring 430, and a secondsupport ring 432. The lamp housing 402 includes a lamp 404, a light-port406, a top 408, and a bottom 410.

In the lighting system 400, the lamp 404 is within the lamp housing 402.The light-port 406 is within the top 408. The infrared absorbing filter414, the optical filter 416, and the second lens 418 are located atpositions above the top 408. The first lens 412 is within the light-port406. The infrared absorbing filter 414 is connected to the light-port406. The optical filter 416 is connected to the infrared absorbingfilter 414. The second lens 418 is supported by the first support ring430. Support for the second lens 418 by the first support ring 430 mayinclude the second lens 418 being on the first support ring 430. Thefirst support ring 430 is removably attached to the ring stand 426. Thesecond lens is located a distance 420 from the optical filter 416. Thecell culture plate 422 is supported by the second support ring 432.Support for the cell culture plate 422 by the second support ring 432may include the cell culture plate 422 being on the second support ring432. The second support ring 432 is removably attached to the ring stand426. The cell culture plate 422 is located a distance 424 from theoptical filter 416. The base 428 is connected to the ring stand 426. Thefirst support ring 430 may be slid up the ring stand 426 to adjust thedistance 420 between the optical filter 414 and the second lens 418. Thefirst support ring 430 may be slid down the ring stand 426 to adjust thedistance 420 between the optical filter 416 and the second lens 418. Thesecond support ring 432 may be slid up the ring stand 426 to adjust thedistance 424 between the optical filter 414 and the cell culture plate422. The second support ring 432 may be slid down the ring stand 426 toadjust the distance 424 between the optical filter 416 and the cellculture plate 422.

Components of the lighting system 400 can be arranged like components ofthe lighting system 100 illustrated in FIG. 1. For example, the lamphousing 402 can be arranged like the lamp housing 102, the lamp 404 canbe arranged like the lamp 104, the light-port 406 can be arranged likethe light-port 106, the first lens 412 can be arranged like the firstlens 108, the infrared absorbing filter 414 can be arranged like theinfrared absorbing filter 110, the optical filter 416 can be arrangedlike the optical filter 114, the second lens 418 can be arranged likethe second lens 116, and the cell culture plate 422 can be arranged likethe cell culture plate 120, except that the cell culture plate 422comprises multiple wells each with a clear bottom but otherwise blackopaque.

In operation, the cell culture plate 422, as well as the contents of thecell culture plate 422, is irradiated with light from the lamp 404.First, broad spectrum light from the lamp 404 exits the lamp 404 bypassing through the light-port 406.

After the broad spectrum light exits the lamp housing 402 by passingthrough light-port 406, the broad spectrum light is collimated by thefirst lens 412.

After the broad spectrum light is collimated by the first lens 412, thecollimated broad spectrum light reaches the infrared absorbing filter414. The infrared absorbing filter 414 absorbs infrared light of thecollimated broad spectrum light. The infrared absorbing filter 414 alsopasses a first portion of the collimated broad spectrum light to theoptical filter 416.

After the first portion of the collimated broad spectrum light is passedby the infrared absorbing filter 414 to the optical filter 416, theoptical filter 416 passes a second portion of the collimated broadspectrum light to the second lens 418. As an example, the light passedto the second lens 318 may be collimated light within the range of 400nm to 630 nm (i.e., no infrared or ultraviolet light). Narrowerband-pass filters can be used to reduce the range.

After the second portion of the broad spectrum light is passed by theoptical filter 416 to the second lens 418, the second lens 418 dispersesthe second portion of the collimated broad spectrum light to the cellculture plate 422.

The distance 420 between the optical filter 416 and the second lens 418and the distance 424 between the optical filter 416 and cell cultureplate 422 may be selected to uniformly irradiate the cell culture plate422. “Uniformly irradiate” and “uniform irradiation” refers to providinglight dispersed by the second lens 416 to the entire area of the cellculture plate 422 or to the entire bottom area of the cell culture plate422.

The distance 420 between the optical filter 416 and the second lens 418to uniformly irradiate the cell culture plate 422 may be 13.6 cm. Otherdistances between the optical filter 416 and the second lens 418 touniformly irradiate the cell culture plate 422 are possible. Thedistance 420 can be a distance between portions of optical filter 416and second lens 418 that are nearest each other. With respect to theorientation of lighting system 400 in FIG. 4, those portions can includean upper side of optical filter 416 and a lower side of second lens 418.Alternatively, the distance 420 between the optical filter 416 and thesecond lens 418 may be a distance between a vertical center point of theoptical filter 416 and a vertical center point of the second lens 418.Other examples of specifying the distance 420 between the optical filter416 and the second lens 418 are possible.

The distance 424 between the optical filter 416 and the cell cultureplate 422 to uniformly irradiate the cell culture plate 422 can bespecified in various ways. In one respect, the distance 424 can bespecified as a distance from a highest point of the optical filter 416to the external surface of the bottom area of the cell culture plate422. In that regard, for a first case, the distance 424 can be 95.5 cm.In another respect, the distance 424 can be specified as a distance fromthe highest point of optical filter 416 to the top area of the cellculture plate 422. In accordance with the first case referred to above,if the vertical wall (the distance between the top area of cell cultureplate 422 and the external surface of the bottom area of the cellculture plate 422) has a height of 1.42 cm, then the distance can be94.08 cm. In another respect, the distance 424 between the opticalfilter 416 and the cell culture plate 422 may be specified as a distancebetween a closest point of the optical filter 416 and a closest point ofthe cell culture plate 422. In yet another respect, the distance 424between the optical filter 416 and the cell culture plate 422 may bespecified as a distance between a vertical center point of the opticalfilter 416 and a vertical center point of the cell culture plate 422.Other examples of specifying the distance 424 between the optical filter416 and the cell culture plate 422 are possible.

In an alternative embodiment, the first lens 412 is outside of thelight-port 406. With this arrangement, the light-port 406 may beconnected to the first lens 412, and the first lens 412 may be connectedto the infrared absorbing filter 414. Alternatively, the light-port 406may be connected to the infrared absorbing filter 414, and the infraredabsorbing filter 414 may be connected to the first lens 412.

In another embodiment, the light-port 406 may be connected to theinfrared absorbing filter 414. With this arrangement, the infraredabsorbing filter 414 is connected to the first lens 412, and the firstlens 412 is connected to the optical filter 416.

In yet another embodiment, the lamp housing 402, the first lens 412, theinfrared absorbing filter 414, the optical filter 416, and the secondlens 418 are each positioned on a ledge (not shown) above a table (notshown). With this arrangement, the cell culture plate 422 may then bepositioned on a temperature controlling device (not shown) located onthe table. The temperature controlling device can be arranged in variousconfigurations. For instance, the temperature controlling device may bea heating device or a cooling device. The temperature controlling devicemay maintain the cell culture plate 422, as well as the contents of thecell culture plate 422, at a regulated temperature.

For simplicity of the block diagrams of FIGS. 1-4, the means forconnecting optical filters 114, 214, 316, and 416 to reflectors 112 and212 and to infrared absorbing filters 314 and 414, respectively are notshown. As an example, those means can be similar to the means shown inFIG. 5 to connect an infrared blocking filter 526 to a reflector 524.

FIGS. 5-7 illustrate another embodiment of a lighting system 500 andcomponents thereof. The lighting system 500 includes a lamp housing 502,a first lens (not shown), an infrared absorbing liquid filter 516, aninlet cooling tube 518, an outlet cooling tube 520, a supporting member521, a pump 522, a cooling system 523, a reflector 524, an infraredblocking filter 526, a short pass filter 528, a first dispersing lens530, a first dispersing lens holder 566, a second dispersing lens 532, asecond dispersing lens holder 568, an adjustment rod 533, a firstadjustment spacer 534, a second adjustment spacer 535, a shelf 540, alens slider 542, and a cell culture plate (not shown). The lamp housing502 includes a lamp (not shown), a mirror (not shown), a light-port (notshown), a top 508, a bottom 510, and a wall 512. The shelf 540 includesa shelf top 546, a shelf riser 548, a first parallel adjustment slot550, a first parallel adjustment fastener 551, a second paralleladjustment slot 552, a second parallel adjustment fastener 553, a secondhole 560 for passing light, a first lens slider attachment 561, a secondlens slider attachment 562, a third lens slider attachment 563, a fourthlens slider attachment 564, and a race 565. The lens slider 542 includesa first hole 544 for passing light, a first perpendicular adjustmentslot 554, a first perpendicular adjustment fastener 555, a secondperpendicular adjustment fastener 556, a second perpendicular adjustmentslot 557, a third perpendicular adjustment fastener 558, and a fourthperpendicular adjustment fastener 559.

In the lighting system 500, the lamp is within the lamp housing like thelamps in the lighting systems illustrated in FIGS. 1-4. The light-portis within the lamp housing 502 like the light-ports in the lightingsystems illustrated in FIGS. 1-4. The mirror can be arranged like themirror 205 of the lighting system 200 illustrated in FIG. 2. The firstlens is within the light-port like the first lenses in the lightingsystems illustrated in FIGS. 1-4. The first lens, the infrared absorbingliquid filter 516, the reflector 524, the infrared blocking filter 526,and the short pass filter 528 are each located at positions between thetop 508 and the bottom 510. The bottom 510 can be metal. The cellculture plate can be arranged like the cell culture plate 120 in thelighting system 100 illustrated in FIG. 1.

Additionally, in the lighting system 500, the infrared absorbing liquidfilter 516 is connected to the light-port like the infrared absorbingfilters are connected to the light-ports in the lighting systemsillustrated in FIGS. 1-4. The infrared absorbing liquid filter 516 isconnected to the inlet cooling tube 518. The inlet cooling tube 518 issupported by the supporting member 521. The inlet cooling tube 518 isconnected to the pump 522. The pump 522 is connected to the coolingsystem 523. The connection can be in-line. The infrared absorbing liquidfilter 516 is connected to the outlet cooling tube 520. The outletcooling tube 520 is supported by the supporting member 521. The outletcooling tube 520 is connected to the pump 522.

Additionally, in the lighting system 500, the reflector 524 is connectedto the infrared absorbing liquid filter 516. The infrared blockingfilter 526 is connected to the reflector 524. The short pass filter 528is connected to the infrared blocking filter 526. The first dispersinglens 530 is located a distance from the short pass filter 528 like thesecond lenses are located a distance from the optical filters in thelighting systems illustrated in FIGS. 1-4. The first dispersing lens 530is supported by the first dispersing lens holder 566. The firstdispersing lens 530 is adjacent to the second dispersing lens 532 with agap between the first dispersing lens 530 and the second dispersing lens532. The gap between the first dispersing lens 530 and the seconddispersing lens 532 is adjustable by the adjustment rod 533, the firstadjustment spacer 534, and the second adjustment spacer 535. Twoadjustment rods 533 each with the first adjustment spacer 534 and thesecond adjustment spacer 535 are shown in FIG. 5 and can be sufficientto adjust the gap between the first dispersing lens 530 and the seconddispersing lens 532. In some embodiments, three or more adjustment rods533 each with the first adjustment spacer 534 and the second adjustmentspacer 535 equally spaced around the first dispersing lens 530 and thesecond dispersing lens 532 are possible. The second dispersing lens 532is supported by the second dispersing lens holder 568. The seconddispersing lens holder 568 is removably attached to the lens slider 542.More specifically, the second dispersing lens holder 568 has a flange(not shown) that rests on the lens slider 542. A portion of the seconddispersing lens holder 568 can be positioned within the first hole 544for passing light or within the first hole 544 and the second hole 560for passing light. Alternatively, a portion of the second dispersinglens holder 568 can extend beyond the first hole 544 for passing lightand the second hole 560 for passing light. The lens slider 542 isremovably attached to shelf top 546. The shelf top 546 is located in adirection perpendicular 536 to the wall 512. The shelf riser 548 islocated in a direction parallel 538 to the wall 512.

Additionally, in the lighting system 500, a position of the firstdispersing lens 530 and the second dispersing lens 532 in the directionperpendicular 536 to the wall 512 is adjustable by the firstperpendicular adjustment slot 554, the first perpendicular adjustmentfastener 555, the second perpendicular adjustment fastener 556, thesecond perpendicular adjustment slot 557, the third perpendicularadjustment fastener 558, and the fourth perpendicular adjustmentfastener 559. A position of the first dispersing lens 530 and the seconddispersing lens 532 in the direction parallel 538 to the wall 512 isadjustable by the first parallel adjustment slot 550, the first paralleladjustment fastener 551, the second parallel adjustment slot 552, andthe second parallel adjustment fastener 553.

In operation, the cell culture plate, as well as the contents of thecell culture plate, is irradiated with light from the lamp. The lamphousing 502 may be Oriel® Instruments Model No. 66921 sold by NewportCorporation. First, broad spectrum light from the lamp exits the lamp bypassing through the light-port. The lamp may be a 1000 watt xenon shortarc lamp. The 1000 watt xenon short arc lamp may be Oriel® InstrumentsModel No. 6271 sold by Newport Corporation. The broad spectrum light maybe reflected by the mirror to the light-port. With this arrangement, themirror may increase the power of the broad spectrum light passingthrough the light-port. Additionally, the mirror may have two or moreadjustments (not shown) to allow the mirror to move in perpendicularplanes. The two or more adjustments can be used to optimize the broadspectrum light for irradiating the cell culture plate. For instance, thetwo or more adjustments can be used to center the broad spectrum lightthrough the first lens. As another example, the two or more adjustmentscan be used to avoid passing a most intense portion of the broadspectrum light through an arc of one or more electrodes associated withthe lamp. The lamp housing 502 may be sealed so that the broad spectrumlight from the lamp exits the lamp housing 502 only through thelight-port. Alternatively, the lamp housing 502 may be sealed so thatless than 1% of the broad spectrum light from the lamp exits the lamphousing 502 other than through the light-port.

After the broad spectrum light exits the lamp housing 502 by passingthrough the light-port, the broad spectrum light is collimated by thefirst lens. The first lens can be arranged in various configurations. Ina first configuration, the first lens may be a 50 mm focal lengthcondenser lens. In that first configuration or in another configuration,the first lens may be a Fresnel lens. Moreover, in that firstconfiguration or in another configuration, the first lens may have adiameter of 2 or 3 in. Other examples of the diameter of the first lensare possible. In a second example configuration, the first lens cancomprise two or more lenses. In accordance with one or more of theseexample embodiments of the first lens, the first lens can comprise alens holder configured for supporting a lens, such as a glass lens, andfor connecting the first lens to another component of the lightingsystem 500, such as infrared absorbing liquid filter 516, thelight-port, the reflector 524, or a spacer.

After the broad spectrum light is collimated by the first lens, thecollimated broad spectrum light reaches the infrared absorbing liquidfilter 516. The infrared absorbing liquid filter 516 may be Oriel®Instruments Model No. 6123 sold by Newport Corporation. The infraredabsorbing liquid filter 516 absorbs infrared light of the collimatedbroad spectrum light. The infrared absorbing liquid filter 516 mayabsorb between 90% and 100%, inclusive, of the infrared light of thecollimated broad spectrum light. Other infrared absorbing ranges of theinfrared absorbing liquid filter 516 are possible. The temperature ofthe infrared absorbing liquid filter 516 may be controlled by pumping acooling fluid to a jacket (not shown) of the infrared absorbing liquidfilter 516 at a flow rate through the inlet cooling tube 518 and theoutlet cooling tube 520 using the pump 522. The cooling fluid may beice-cold water. The cooling fluid may be cooled by the cooling system523. The flow rate may be 6 milliliters per minute (mL/min). The pump522 may be a peristaltic pump. The infrared absorbing liquid filter 516also passes a first portion of the collimated broad spectrum light tothe reflector 524.

After the first portion of the collimated broad spectrum light is passedby the infrared absorbing liquid filter 516 to the reflector 524, thereflector 524 reflects the first portion of the collimated broadspectrum light. The first portion of the collimated broad spectrum lightthen propagates to the infrared blocking filter 526. Alternatively, thereflector 524 may reflect only a portion of the first portion of thecollimated broad spectrum light, and the portion of the first portion ofthe collimated broad spectrum light then propagates to the infraredblocking filter 526. The reflector 524 can be arranged in a variety ofconfigurations. In a first configuration, the reflector 524 may be abeam turning dichroic mirror. The reflector 524 may be Oriel®Instruments Model No. 66229 sold by Newport Corporation. In that firstconfiguration or another configuration, the reflector 524 may have a 2.0in diameter. Other diameters for the reflector 524 are possible. In asecond configuration, the reflector 524 may be two or more beam turningdichroic mirrors. The first portion of the collimated broad spectrumlight that is reflected by the reflector 524 to the infrared blockingfilter 526 may have a wavelength in a given range. For example, thecollimated broad spectrum light may have a given range of 400 nm to 700nm. Other given ranges are possible. In some embodiments, the reflector524 absorbs at least a portion of infrared light or at least a portionof ultraviolet light of the collimated broad spectrum light. With thisarrangement, the reflector 524 functions like an optical filter bypassing only visible light to the infrared blocking filter 526.

After the first portion of the collimated broad spectrum lightpropagates to the infrared blocking filter 526, the infrared blockingfilter 526 absorbs residual infrared light of the first portion of thecollimated broad spectrum light. The infrared blocking filter 526 alsopasses a filtered first portion of the collimated broad spectrum lightto the short pass filter 528. The infrared blocking filter 526 may be a50% infrared blocking filter. The infrared blocking filter 526 may beOriel® Instruments Model No. 59043 sold by Newport Corporation.

After the filtered first portion of the collimated broad spectrum lightis passed by the infrared absorbing filter 526 to the short pass filter528, the short pass filter 528 passes a second portion of the collimatedbroad spectrum light to the first dispersing lens 530. The short passfilter 528 may have a cut-off of 650 nm. If reflector 524 passeswavelengths greater than 650 nm, short pass filter 528 will block suchwavelengths. If reflector 524 does not pass any wavelength greater than650 nm, then short pass filter may not block any wavelength. The cut-offspecified for short pass filter 528 can be less than, equal to orgreater than highest wavelengths passed by short pass filter 528.Additionally, the cut-off specified for short pass filter 528 can beless than the highest wavelength passed by the reflector 524. The shortpass filter 528 may be Andover Corporation Part No. 650-FL07-50.Different ranges can be passed to the first dispersing lens 530depending on what light is reflected by the reflector 524 and passed bythe short pass filter 528. For example, the second portion of thecollimated broad spectrum light may have a given range of 400 nm to 630nm. As another example, the range of wavelengths can be any variety ofranges, such as between about 595 nm and about 645 nm, between about 620nm and about 640 nm, between about 615 nm and about 645 nm, or betweenabout 610 nm and about 650 nm. Narrower band-pass filters can be used toreduce the range. The range of wavelengths can be centered on a givenwavelength, such as 630 nm. A different center wavelength can beselected by changing components of the lighting system 500, such as thereflector 524 and the short pass filter 528. The first dispersing lens530 can be arranged in a variety of configurations. In a firstconfiguration, the first dispersing lens 530 may have a diameter of 3in. Other diameters of the first dispersing lens 530 are possible. Inthat first configuration or another configuration, the first dispersinglens 530 may have an effective focal length of 200 mm. Other effectivefocal lengths of the first dispersing lens 530 are possible.

After the second portion of the collimated broad spectrum light ispassed by the short pass filter 528 to the first dispersing lens 530,the first dispersing lens 530 passes the second portion of the broadspectrum light to the second dispersing lens 532. The second dispersinglens 532 can be arranged in a variety of configurations. In a firstconfiguration, the second dispersing lens 532 may have a diameter of 3in. Other diameters of the second dispersing lens 532 are possible. Inthat first configuration or another configuration, the second dispersinglens 532 may have an effective focal length of 200 mm. Other effectivefocal lengths of the second dispersing lens 532 are possible.

After the second portion of the collimated broad spectrum light ispassed by the first dispersing lens 530 to the second dispersing lens532, the second dispersing lens 532 disperses the second portion of thecollimated broad spectrum light to the cell culture plate. The seconddispersing lens 532 disperses the second portion of the collimated broadspectrum light to the cell culture plate through the first hole 544 forpassing light and the second hole 560 for passing light. At least aportion of the first hole 544 for passing light may be above or below atleast a portion of the second hole 560 for passing light.

The distance between the short pass filter 528 and the first dispersinglens 530, the gap between the first dispersing lens 530 and the seconddispersing lens 532, and the distance between the short pass filter 528and the cell culture plate may be selected to uniformly irradiate thecell culture plate. “Uniformly irradiate” and “uniform irradiation”refers to providing light dispersed by the second dispersing lens 532 tothe entire area of the cell culture plate or to the entire bottom areaof the cell culture plate.

The distance between the short pass filter 528 and the first dispersinglens 530 to uniformly irradiate the cell culture plate may be 13.6 cm.Other distances between the short pass filter 528 and the firstdispersing lens 530 to uniformly irradiate the cell culture plate arepossible. The distance can be a distance between portions of short passfilter 528 and the first dispersing lens 530 that are nearest eachother. With respect to the orientation of lighting system 500 in FIG. 5,those portions can include a lower side of short pass filter 528 and anupper side of the first dispersing lens 530. Alternatively, the distancebetween the short pass filter 528 and the first dispersing lens 530 maybe a distance between a vertical center point of the short pass filter528 and a vertical center point of the first dispersing lens 530. Otherexamples of specifying the distance between the short pass filter 528and the first dispersing lens 530 are possible.

The gap between the first dispersing lens 530 and the second dispersinglens 532 to uniformly irradiate the cell culture plate with the secondportion of the broad spectrum light may be 3 mm. Other gaps between thefirst dispersing lens 530 and the second dispersing lens 532 arepossible. For instance, the gap between the first dispersing lens 530and the second dispersing lens 532 may be within the range of 2 mm to 4mm, inclusive. The gap between the first dispersing lens 530 and thesecond dispersing lens 532 may be a distance between a closest point ofthe first dispersing lens 530 and a closest point of the seconddispersing lens 532. Alternatively, the gap between the first dispersinglens 530 and the second dispersing lens 532 may be a distance between avertical center point of the first dispersing lens 530 and a verticalcenter point of the second dispersing lens 532. Other examples ofspecifying the gap between the first dispersing lens 530 and the seconddispersing lens 532 are possible.

The distance between the short pass filter 528 and the cell cultureplate to uniformly irradiate the cell culture plate can be specified invarious ways. In one respect, the distance can be specified as adistance from a highest point of short pass filter 528 to the externalsurface of the bottom area of the cell culture plate. In that regard,for a first case, the distance can be 95.5 cm. In another respect, thedistance can be specified as a distance from the highest point of shortpass filter 528 to the top area of the cell culture plate. In accordancewith the first case referred to above, if the vertical wall (thedistance between the top area of cell culture plate and the externalsurface of the bottom area of the cell culture plate) has a height of1.42 cm, then the distance can be 94.08 cm. In another respect, thedistance between the short pass filter 528 and the cell culture platemay be specified as a distance between a closest point of the short passfilter 528 and a closest point of the cell culture plate. In yet anotherrespect, the distance between the short pass filter 528 and the cellculture plate may be specified as a distance between a vertical centerpoint of the short pass filter 528 and a vertical center point of thecell culture plate. Other examples of specifying the distance betweenthe short pass filter 528 and the cell culture plate are possible.

As noted above, the position of the first dispersing lens 530 and thesecond dispersing lens 532 in the direction perpendicular 536 to thewall 512 is adjustable by the first perpendicular adjustment slot 554,the first perpendicular adjustment fastener 555, the secondperpendicular adjustment fastener 556, the second perpendicularadjustment slot 557, the third perpendicular adjustment fastener 558,and the fourth perpendicular adjustment fastener 559. With thisarrangement, the position of the first dispersing lens 530 and thesecond dispersing lens 532 in the direction perpendicular 536 to thewall 512 may be adjusted to align a center of the first dispersing lens530 with a center of the short pass filter 528. The position of thefirst dispersing lens 530 and the second dispersing lens 532 in adirection perpendicular 536 to the wall 512 may be adjusted by adjustinga location of the first perpendicular adjustment fastener 555 in thefirst perpendicular adjustment slot 554, adjusting a location of thesecond perpendicular adjustment fastener 556 in the first perpendicularadjustment slot 554, adjusting a location of the third perpendicularadjustment fastener 558 in the second perpendicular adjustment slot 557,and adjusting a location of the fourth perpendicular adjustment fastener559 in the second perpendicular adjustment slot 557. The perpendicularadjustment fasteners (555, 556, 558, 559) may each be a bolt. Theperpendicular adjustment fasteners (555, 556, 558, 559) may be otherfasteners, such as screws. Adjusting the location of one of theperpendicular adjustment fasteners (555, 556, 558, 559) in one of theperpendicular adjustment slots (554, 557) may refer to translating theperpendicular adjustment fasteners (555, 556, 558, 559) within theperpendicular adjustment slots (554, 557).

As noted above, the gap between the first dispersing lens 530 and thesecond dispersing lens 532 is adjustable by the adjustment rod 533, thefirst adjustment spacer 534, and the second adjustment spacer 535. Thegap between the first dispersing lens 530 and the second dispersing lens532 may be adjusted by adjusting at least one position of the firstadjustment spacer 534 and the second adjustment spacer 535 along theadjustment rod 533. The adjustment rod 533 may be threaded to engagewith the first adjustment spacer 534 and the second adjustment spacer535. The first adjustment spacer 534 may be a washer. Similarly, thesecond adjustment spacer 535 may be a washer.

As noted above, the position of the first dispersing lens 530 and thesecond dispersing lens 532 in the direction parallel 538 to the wall 512is adjustable by the first parallel adjustment slot 550, the firstparallel adjustment fastener 551, the second parallel adjustment slot552, and the second parallel adjustment fastener 553. With thisarrangement, the position of the first dispersing lens 530 and thesecond dispersing lens 532 in a direction parallel 538 to the wall 512may be adjusted to align a center of the first dispersing lens 530 witha center of the short pass filter 528. The position of the firstdispersing lens 530 and the second dispersing lens 532 in a directionparallel 538 to the wall 512 may be adjusted by adjusting a location ofthe first parallel adjustment fastener 551 in the first paralleladjustment slot 550 and adjusting a location of the second paralleladjustment fastener 553 in the second parallel adjustment slot 552. Thefirst parallel adjustment fastener 551 and the second paralleladjustment fastener 553 may each be a screw. The first paralleladjustment fastener 551 and the second parallel adjustment fastener 553may be other fasteners, such as a bolt with a corresponding nut.Adjusting the location of the first parallel adjustment fastener 551 inthe first parallel adjustment slot 550 may refer to translating thefirst parallel adjustment fastener 551 within the first paralleladjustment slot 550. Similarly, adjusting the location of the secondparallel adjustment fastener 553 in the second parallel adjustment slot552 may refer to translating the second parallel adjustment fastener 553within the second parallel adjustment slot 552.

As noted above, the lens slider 542 is removably attached to the shelftop 546. The lens slider may be removably attached to the shelf top byattaching the first perpendicular adjustment fastener 555 to the firstlens slider attachment 561, attaching the second perpendicularadjustment fastener 556 to the second lens slider attachment 562,attaching the third perpendicular adjustment fastener 558 to the thirdlens slider attachment 563, and attaching the fourth perpendicularadjustment fastener 559 to the fourth lens slider attachment 564. Thelens slider attachments (561, 562, 563, 564) may each be a hole or aslot with a corresponding nut (not shown). The lens slider attachments(561, 562, 563, 564) may be other attachments, such as a threaded hole.Other connections of the lens slider 542 to the shelf 540 are possible.

Although FIG. 7 shows the first lens slider attachment 561, the secondlens slider attachment 562, the third lens slider attachment 563, andthe fourth lens slider attachment 564 as being circular, a personskilled in the art will understand that 561, 562, 563, and 564 can beelongated slots to allow for additional adjustment of the firstdispersing lens 530 and the second dispersing lens 532.

In an alternative embodiment, the reflector 524 is connected to theshort pass filter 528. With this arrangement, the short pass filter 528is connected to the infrared blocking filter 526.

In another embodiment, the light-port is connected to the infraredabsorbing liquid filter 516. With this arrangement, the infraredabsorbing liquid filter 516 is connected to the first lens, and thefirst lens is connected to the reflector 524.

In another embodiment, the first lens is outside of the light-port. Withthis arrangement, the light-port may be connected to the first lens, thefirst lens may be connected to the infrared absorbing liquid filter 516,and the infrared absorbing liquid filter 516 may be connected to thereflector 524. Alternatively, the light-port may be connected to theinfrared absorbing liquid filter 516, the infrared absorbing liquidfilter 516 may be connected to the first lens, and the first lens may beconnected to the reflector 524. In accordance with these embodiments,the infrared blocking filter 526 or the short pass filter 528 should bedownstream of the infrared absorbing liquid filter 516 or the reflector524 to avoid the heat of the light cracking the infrared blocking filter526 or the short pass filter 528.

In another embodiment, the lamp housing 502, the first lens, theinfrared absorbing liquid filter 516, the reflector 524, the infraredblocking filter 526, the short pass filter 528, the first dispersinglens 530, and the second dispersing lens 532 are each positioned on aledge (not shown) above a table (not shown). With this arrangement, thecell culture plate may then positioned on a temperature controllingdevice (not shown) located on the table. The temperature controllingdevice can be arranged in various configurations. For instance, thetemperature controlling device may be a heating device or a coolingdevice. The temperature control device may maintain the cell cultureplate, as well as the contents of the cell culture plate, at a regulatedtemperature.

In yet another embodiment, the reflector 524 is inverted. With thisarrangement, the reflector 524 reflects the first portion of thecollimated broad spectrum light upward. As such, the infrared blockingfilter 526, the short pass filter 528, the first dispersing lens 530,the second dispersing lens 532, and the cell culture plate are eachlocated above the reflector 524. In accordance with this embodiment,uniform irradiation of the cell culture plate comprises providing lightdispersed by the second dispersing lens 532 to the entire bottom area ofthe cell culture plate.

FIGS. 8A-C and 9 illustrate another embodiment of a lighting systemgenerally designated 800 and components thereof. The lighting system 800includes a lamp housing 802, a first lens (not shown), an infraredabsorbing liquid filter 816, an inlet cooling tube 818, and outletcooling tube 820, a supporting member 821, a pump 822, a cooling system823, a reflector 824, an infrared blocking filter 826, a short passfilter 828, a first plano convex lens 830, a first plano convex lensholder 870, a second plano convex lens 832, a second plano convex lensholder 872, an adjustment rod 841, a first adjustment spacer 842, asecond adjustment spacer 843, a base wall 848, a shelf 850, a lensslider 852, and cell culture plate (not shown). The lamp housing 802includes a lamp (not shown), a mirror (not shown), a light-port (notshown), a top 808, a bottom 810, and a wall 812. The first plano convexlens 830 includes a first convex side 836 and a first plane side (notshown). The second plano convex lens 832 includes a second convex side840 and a second plane side (not shown). The shelf 850 includes a shelftop 854, a shelf riser 856, a first parallel adjustment slot 858, afirst parallel adjustment fastener 859, a second parallel adjustmentslot 860, a second parallel adjustment fastener 861, a second hole 868for passing light, a race (not shown), a first lens slider attachment(not shown), a second lens slider attachment (not shown), a third lensslider attachment (not shown), and a fourth lens slider attachment (notshown). The lens slider 852 includes a first perpendicular adjustmentslot 862, a first perpendicular adjustment fastener 863, a secondperpendicular adjustment fastener 864, a second perpendicular adjustmentslot 865, a third perpendicular adjustment fastener 866, a fourthperpendicular adjustment fastener 867, and a first hole (not shown) forpassing light.

In the lighting system 800, the lamp is within the lamp housing 802 likethe lamps in the lighting systems illustrated in FIGS. 1-4. Thelight-port is within the lamp housing 802 like the light-ports in thelighting systems illustrated in FIGS. 1-4. The first lens is within thelight-port like the first lenses in the lighting systems illustrated inFIGS. 1-4. The first lens, the infrared absorbing liquid filter 816, thereflector 824, the infrared blocking filter 826, and the short passfilter 828 are each located at positions between the top 808 and thebottom 810. The bottom 810 can be metal.

Additionally, in the lighting system 800, the infrared absorbing liquidfilter 816 is connected to the light-port like the infrared absorbingfilters are connected to the light-ports in the lighting systemsillustrated in FIGS. 1-4. The infrared absorbing liquid filter 816 isconnected to the inlet cooling tube 818. The inlet cooling tube 818 issupported by the supporting member 821. The inlet cooling tube 818 isconnected to the pump 822. The pump 822 is connected to the coolingsystem 823. The connection can be in-line. The infrared absorbing liquidfilter 816 is connected to the outlet cooling tube 820. The outletcooling tube 820 is supported by the supporting member 821. The outletcooling tube 820 is connected to the pump 822.

Additionally, in the lighting system 800, the reflector 824 is connectedto the infrared absorbing liquid filter 816. The infrared blockingfilter 826 is connected to the reflector 824. The short pass filter 828is connected to the infrared blocking filter 826. The first plano convexlens 830 is located a distance from the infrared blocking filter 826like the second lenses are located a distance from the optical filtersin the lighting systems illustrated in FIGS. 1-4. The first plano convexlens 830 is supported by the first plano convex lens holder 870. Thefirst plano convex lens 830 is adjacent to the second plano convex lens832 with a gap between the first convex side 836 and the second convexside 840. The gap between the first convex side 836 and the secondconvex side 840 is adjustable by the adjustment rod 841, the firstadjustment spacer 842, and the second adjustment spacer 843. Twoadjustment rods 841 each with the first adjustment spacer 842 and thesecond adjustment spacer 843 are shown in FIG. 8A and can be sufficientto adjust the gap between the first convex side 836 and the secondconvex side 840. In some embodiments, three or more adjustment rods 841each with the first adjustment spacer 842 and the second adjustmentspacer 843 equally spaced around the first plano convex lens 830 and thesecond plano convex lens 832 are possible. The second plano convex lens832 is supported by the second plano convex lens holder 872. The secondplano convex lens holder 872 is removably attached to the lens slider852. More specifically, the second plano convex lens holder 872comprises a flange (not shown) that rests on the lens slider 852. Aportion of the second dispersing lens holder 872 can be positionedwithin the first hole for passing light or within the first hole and thesecond hole 868 for passing light. Alternatively, a portion of thesecond plano convex lens holder 872 can extend beyond the first hole forpassing light and the second hole 868 for passing light. The lens slider852 is removably attached to the shelf top 854. The shelf 850 isconnected to the base wall 848. The shelf riser 856 is located in adirection parallel 844 to the wall 812. The shelf top 854 is located ina direction perpendicular 846 to the wall 812. A position of the firstplano convex lens 830 and the second plano convex lens 832 is adjustablein the direction parallel 844 to the wall 812. A position of the firstplano convex lens 830 and the second plano convex lens 832 is adjustablein the direction perpendicular 846 to the wall 812.

Components in the lighting system 800 can be arranged like thecomponents in the lighting system 500 illustrated in FIGS. 5-7. Forexample, the lamp housing 802 can be arranged like the lamp housing 502,the lamp can be arranged like the lamp, the light-port can be arrangedlike the light-port, the first lens can be arranged like the first lensof the lighting system 500, the mirror can be arranged like the mirrorof the lighting system 500, the infrared absorbing liquid filter 816 canbe arranged like the infrared absorbing liquid filter 516, the inletcooling tube 818 can be arranged like the inlet cooling tube 518, theoutlet cooling tube 820 can be arranged like the outlet cooling tube520, the pump 822 can be arranged like the pump 522, the cooling system823 can be arranged like the cooling system 523, the reflector 824 canbe arranged like the reflector 524, the infrared blocking filter 826 canbe arranged like the infrared blocking filter 526, the short pass filter828 can be arranged like the short pass filter 528, the shelf 850 can bearranged like the shelf 540, the shelf top 854 can be arranged like theshelf top 546, the shelf riser 856 can be arranged like the shelf riser548, the first parallel adjustment slot 858 can be arranged like thefirst parallel adjustment slot 550, the first parallel adjustmentfastener 859 can be arranged like the first parallel adjustment fastener551, the second parallel adjustment slot 860 can be arranged like thesecond parallel adjustment slot 552, the second parallel adjustmentfastener 861 can be arranged like the second parallel adjustmentfastener 553, the second hole 868 for passing light can be arranged likethe second hole 560 for passing light, the first lens slider attachmentcan be arranged like the first lens slider attachment 561, the secondlens slider attachment can be arranged like the second lens sliderattachment 562, the third lens slider attachment can be arranged likethe third lens slider attachment 563, the fourth lens slider attachmentcan be arranged like the fourth lens slider attachment 564, the race canbe arranged like the race 565, the lens slider 852 can be arranged likethe lens slider 542, the first perpendicular adjustment slot 862 can bearranged like the first perpendicular adjustment slot 554, the firstperpendicular adjustment fastener 863 can be arranged like the firstperpendicular adjustment fastener 555, the second perpendicularadjustment fastener 864 can be arranged like the second perpendicularadjustment fastener 556, the second perpendicular adjustment slot 865can be arranged like the second perpendicular adjustment slot 557, thethird perpendicular adjustment fastener 866 can be arranged like thethird perpendicular adjustment fastener 558, the fourth perpendicularadjustment fastener 867 can be arranged like the fourth perpendicularadjustment fastener 559, and the first hole for passing light can bearranged like the first hole 544 for passing light. Additionally, thecell culture plate can be arranged like the cell culture plate 120 inthe lighting system 100 illustrated in FIG. 1.

In operation, the cell culture plate, as well as the contents of thecell culture plate, is irradiated with light from the lamp. First, broadspectrum light from the lamp exits the lamp by passing through thelight-port. The broad spectrum light may be reflected by the mirror tothe light-port.

After the broad spectrum light exits the lamp housing 802 by passingthrough the light-port, the broad spectrum light is collimated by thefirst lens.

After the broad spectrum light is collimated by the first lens, thecollimated broad spectrum light reaches the infrared absorbing liquidfilter 816. The infrared absorbing liquid filter 816 absorbs infraredlight of the collimated broad spectrum light. The infrared absorbingliquid filter 816 may absorb between 90% and 100%, inclusive, of theinfrared light of the broad spectrum light. Other infrared absorbingranges of the infrared absorbing liquid filter 816 are possible. Thetemperature of the infrared absorbing liquid filter 816 may becontrolled by pumping a cooling fluid to a jacket (not shown) of theinfrared absorbing liquid filter 816 at a flow rate through the inletcooling tube 818 and the outlet cooling tube 820 using the pump 822. Thecooling fluid may be ice-cold water. The cooling fluid may be cooled bythe cooling system 823. The flow rate may be 6 mL/min. The infraredabsorbing liquid filter 816 also passes a first portion of thecollimated broad spectrum light to the reflector 824.

After the first portion of the collimated broad spectrum light is passedby the infrared absorbing liquid filter 816 to the reflector 824, thereflector 824 reflects the first portion of the collimated broadspectrum light. The first portion of the collimated broad spectrum lightthen propagates to the infrared blocking filter 826. Alternatively, thereflector 824 may reflect only a portion of the first portion of thecollimated broad spectrum light, and the portion of the first portion ofthe collimated broad spectrum light then propagates to the infraredblocking filter 826. The reflector 824 reflects light with the samegiven range of wavelengths as the reflector 524.

After the first portion of the broad spectrum light propagates to theinfrared blocking filter 826, the infrared blocking filter 826 absorbsresidual infrared light of the first portion of the collimated broadspectrum light. The infrared blocking filter 826 also passes a filteredfirst portion of the broad spectrum light to the short pass filter 828.

After the filtered first portion of the collimated broad spectrum lightis passed by the infrared blocking filter 826 to the short pass filter828, the short pass filter 828 passes a second portion of the broadspectrum light to the first plano convex lens 830. The short pass filter828 passes light with the same given range of wavelengths as the shortpass filter 528. The first plano convex lens 830 can be arranged in avariety of configurations. In a first configuration, the first planoconvex lens 830 may have a diameter of 3 in. Other diameters of thefirst plano convex lens 830 are possible. In that first configuration oranother configuration, the first plano convex lens 830 may have aneffective focal length of 200 mm. Other effective focal lengths of thefirst plano convex lens 830 are possible. The first plano convex lens830 may be Newport Corporation Part No. KPX229.

After the second portion of the collimated broad spectrum light ispassed by the short pass filter 828 to the first plano convex lens 830,the first plano convex lens 830 passes the second portion of thecollimated broad spectrum light to the second plano convex lens 832. Thesecond plano convex lens 832 can be arranged in a variety ofconfigurations. In a first configuration, the second plano convex lens832 may have a diameter of 3 in. Other diameters of the second planoconvex lens 832 are possible. In that first configuration or anotherconfiguration, the second plano convex lens 832 may have an effectivefocal length of 200 mm. Other effective focal lengths of the secondplano convex lens 832 are possible. The second plano convex lens 832 maybe Newport Corporation Part No. KPX299. The first plano convex lensholder 870 and the second plano convex lens holder 872 may be individualcomponents or a single unit, such as Newport Corporation Part No. 6216.

After the second portion of the collimated broad spectrum light ispassed by the first plano convex lens 830 to the second plano convexlens 832, the second plano convex lens 832 disperses the second portionof the collimated broad spectrum light to the cell culture plate. Thesecond plano convex lens 832 disperses the second portion of thecollimated broad spectrum light to the cell culture plate through thefirst hole for passing light and the second hole 868 for passing light.At least a portion of the first hole for passing light may be above orbelow at least a portion of the second hole 868 for passing light.

The distance between the short pass filter 828 and the first planoconvex lens 830, the gap between the first convex side 836 and thesecond convex side 840, and the distance between the second plano convexlens 832 and the cell culture plate may be selected to uniformlyirradiate the cell culture plate. “Uniformly irradiate” and “uniformirradiation” refers to providing light dispersed by the second planoconvex lens 832 to the entire area of the cell culture plate or to theentire bottom area of the cell culture plate.

The distance between the short pass filter 828 and the first planoconvex lens 830 to uniformly irradiate the cell culture plate may be13.6 cm. Other distances between the short pass filter 828 and the firstplano convex lens 830 to uniformly irradiate the cell culture plate arepossible. The distance can be a distance between portions of short passfilter 828 and the first plano convex lens 830 that are nearest eachother. With respect to the orientation of lighting system 800 in FIGS.8A-C and 9, those portions can include a lower side of short pass filter828 and an upper side of the first plano convex lens 830. Alternatively,the distance between the short pass filter 828 and the first planoconvex lens 830 may be a distance between a vertical center point of theshort pass filter 828 and a vertical center point of the first planoconvex lens 830. Other examples of specifying the distance between theshort pass filter 828 and the first plano convex lens 830 are possible.

The gap between the first convex side 836 and the second convex side 840to uniformly irradiate the cell culture plate with the second portion ofthe broad spectrum light may be 3 mm. Other gaps between the firstconvex side 836 and the second convex side 840 are possible. Forinstance, the gap between the first convex side 836 and the secondconvex side 840 may be within the range of 2 mm to 4 mm, inclusive. Thegap between the first convex side 836 and the second convex side 840 maybe a distance between a closest point of the first convex side 836 and aclosest point of the second convex side 840. Alternatively, the gapbetween the first convex side 836 and the second convex side 840 may bea distance between a vertical center point of the first convex side 836and a vertical center point of the second convex side 840. Otherexamples of specifying the gap between the first convex side 836 and thesecond convex side 840 are possible.

The distance between the short pass filter 828 and the cell cultureplate to uniformly irradiate the cell culture plate can be specified invarious ways. In one respect, the distance can be specified as adistance from a highest point of short pass filter 828 to the externalsurface of the bottom area of the cell culture plate. In that regard,for a first case, the distance can be 95.5 cm. In another respect, thedistance can be specified as a distance from the highest point of shortpass filter 828 to the top area of the cell culture plate. In accordancewith the first case referred to above, if the vertical wall (thedistance between the top area of cell culture plate and the externalsurface of the bottom area of the cell culture plate) has a height of1.42 cm, then the distance can be 94.08 cm. In another respect, thedistance between the short pass filter 828 and the cell culture platemay be specified as a distance between a closest point of the short passfilter 828 and a closest point of the cell culture plate. In yet anotherrespect, the distance between the short pass filter 828 and the cellculture plate may be specified as a distance between a vertical centerpoint of the short pass filter 828 and a vertical center point of thecell culture plate. Other examples of specifying the distance betweenthe short pass filter 828 and the cell culture plate are possible.

As noted above, the position of the first plano convex lens 830 and thesecond plano convex lens 832 in the direction parallel 844 to the wall812 is adjustable by the first parallel adjustment slot 858, the firstparallel adjustment fastener 859, the second parallel adjustment slot860, and the second parallel adjustment fastener 861. With thisarrangement, the position of the first plano convex lens 830 and thesecond plano convex lens 832 in the direction parallel 844 to the wall812 may be adjusted to align a center of the first plano convex lens 830with a center of the short pass filter 828. The position of the firstplano convex lens 830 and the second plano convex lens 832 in thedirection parallel 844 to the wall 812 may be adjusted by adjusting alocation of the first parallel adjustment fastener 859 in the firstparallel adjustment slot 858 and adjusting a location of the secondparallel adjustment fastener 861 in the second parallel adjustment slot860. Adjusting the location of the first parallel adjustment fastener859 in the first parallel adjustment slot 858 may refer to translatingthe first parallel adjustment fastener 859 within the first paralleladjustment slot 858. Similarly, adjusting the location of the secondparallel adjustment fastener 861 in the second parallel adjustment slot860 may refer to translating the second parallel adjustment fastener 861within the second parallel adjustment slot 860.

As noted above, the gap between the first convex side 836 and the secondconvex side 840 is adjustable by the adjustment rod 841, the firstadjustment spacer 842, and the second adjustment spacer 843. The gapbetween the first convex side 836 and the second convex side 840 may beadjusted by adjusting at least one position of the first adjustmentspacer 842 and the second adjustment spacer 843 along the adjustment rod841. The adjustment rod 841 may be threaded to engage with the firstadjustment spacer 842 and the second adjustment spacer 843.

As noted above, the position of the first plano convex lens 830 and thesecond plano convex lens 832 in the direction perpendicular 846 to thewall 812 is adjustable by the first perpendicular adjustment slot 862,the first perpendicular adjustment fastener 863, the secondperpendicular adjustment fastener 864, the second perpendicularadjustment slot 865, the third perpendicular adjustment fastener 866,and the fourth perpendicular adjustment fastener 867. With thisarrangement, the position of the first plano convex lens 830 and thesecond plano convex lens 832 in the direction perpendicular 846 to thewall 812 may be adjusted to align the center of the first plano convexlens 830 with the center of the short pass filter 828. The position ofthe first plano convex lens 830 and the second plano convex lens 832 ina direction perpendicular 846 to the wall 812 may be adjusted byadjusting a location of the first perpendicular adjustment fastener 863in the first perpendicular adjustment slot 862, adjusting a location ofthe second perpendicular adjustment fastener 864 in the firstperpendicular adjustment slot 862, adjusting a location of the thirdperpendicular adjustment fastener 866 in the second perpendicularadjustment slot 865, and adjusting a location of the fourthperpendicular adjustment fastener 867 in the second perpendicularadjustment slot 865. Adjusting the location of one of the perpendicularadjustment fasteners (863, 864, 866, 867) in one of the perpendicularadjustment slots (862, 865) may refer to translating the perpendicularadjustment fasteners (863, 864, 866, 867) within the perpendicularadjustment slots (862, 865).

In an alternative embodiment, the reflector 824 is connected to theshort pass filter 828. With this arrangement, the short pass filter 828is connected to the infrared blocking filter 826.

In another embodiment, the light-port is connected to the infraredabsorbing liquid filter 816. With this arrangement, the infraredabsorbing liquid filter 816 is connected to the first lens, and thefirst lens is connected to the reflector 824.

In another embodiment, the first lens is outside of the light-port. Withthis arrangement, the light-port may be connected to the first lens, thefirst lens may be connected to the infrared absorbing liquid filter 816,and the infrared absorbing liquid filter 816 may be connected to thereflector 824. Alternatively, the light-port may be connected to theinfrared absorbing liquid filter 816, the infrared absorbing liquidfilter 816 may be connected to the first lens, and the first lens may beconnected to the reflector 824. In accordance with these embodiments,the infrared blocking filter 826 or the short pass filter 828 should bedownstream of the infrared absorbing liquid filter 816 or the reflector824 to avoid the heat of the light cracking the infrared blocking filter826 or the short pass filter 828.

In another embodiment, the lamp housing 802, the first lens, theinfrared absorbing liquid filter 816, the reflector 824, the infraredblocking filter 826, the short pass filter 828, the first plano convexlens 830, and the second plano convex lens 832 are each positioned on aledge (not shown) above a table (not shown). With this arrangement, thecell culture plate is then positioned on a temperature controllingdevice (not shown) located on the table. The temperature controllingdevice can be arranged in various configurations. For instance, thetemperature controlling device may be a heating device or a coolingdevice. The temperature controlling device may maintain the cell cultureplate, as well as the contents of the cell culture plate, at a regulatedtemperature.

In yet another embodiment, the reflector 824 is inverted. With thisarrangement, the reflector 824 reflects the first portion of thecollimated broad spectrum light upward. As such, the infrared blockingfilter 826, the short pass filter 828, the first plano convex lens 830,the second plano convex lens 832, and the cell culture plate are eachlocated above the reflector 824. In accordance with this embodiment,uniform irradiation of the cell culture plate comprises providing lightdispersed by the second plano convex lens 832 to the entire bottom areaof the cell culture plate.

For any of the embodiments of the lighting systems described herein, alight meter can measure light, detectable by a probe, between the lampand the cell culture plate. The light meter may be a hand held powermeter, such as Newport Corporation Part No. 1916-R. The probe may be ameter probe or sensor, such as Newport Corporation Part No. 818P-001-12.

2. Example Operation

The lighting system 100 may be used to irradiate contents of a cellculture plate 120. This method is applicable to any of the examplelighting systems discussed above. The cell culture plate may comprise aphotosensitizer.

A method 1000 for studying a photosensitizer is provided as shown inFIG. 10. The method 1000 comprises adding the photosensitizer to aportion of wells on a cell culture plate to form photosensitizer assaywells (1002), the wells comprising carcinoma cells, selecting a firstpredetermined time period (1004), and incubating the photosensitizerassay wells for the first predetermined time period (1006). Thecarcinoma cells may be, for example, A549 human lung carcinoma cells.The method may also be used with any type of cell line, such ascarcinoma, tumor, and normal cell lines. The cell culture plate may bean opaque black plate.

The method 1000 further comprises selecting a predetermined wavelength(1008) and optionally washing the photosensitizer assay wells (1010).The method 1000 also comprises irradiating the photosensitizer assaywells with the lighting system at the predetermined wavelength to formirradiated wells to form uniformly irradiated wells (1012). For example,the photosensitizer assay wells may be irradiated at between about 400nm and about 650 nm. In other embodiments, the photosensitizer assaywells may be irradiated at different wavelengths, for example betweenabout 595 nm and about 645 nm, between about 620 nm and about 640 nm,between about 615 nm and about 645 nm, or between about 610 nm and about650 nm. Depending on the type of photosensitizer used, differentwavelengths may be used. The irradiating step may be standardized suchthat the step is repeated in the same way each time. The lighting system100 discussed in detail above provides for this standardization.

The method 1000 further comprises selecting a second predetermined timeperiod (1014), incubating the irradiated wells for the secondpredetermined time period (1016), and determining percent viability ofthe carcinoma cells contained in the wells (1018).

In some embodiments, the step of washing the photosensitizer assay wells(1010) is mandatory. The remaining portion of the wells may be controlwells. A portion of the wells may contain a reference drug used forcomparison.

The photosensitizer is a porphyrin-based anti-neoplastic agent and theporphyrin-based anti-neoplastic agent may be porfimer sodium.

Examples 1. Overview

Cell passaging of A549 cells is performed using fixed seeding densitiesto reduce assay variation resulting from cell culture conditions. Foreach assay, cells are seeded into sterile, black, tissue-culturedtreated 96-well plates at 10,000 cells per well in a 0.1 mL volume andincubated at 37° C. in an atmosphere of 5% CO₂ overnight. Working invery low light conditions, Photofrin® is reconstituted in normal salineto 2.5 mg/mL and diluted further in culture medium at twice the desiredfinal assay concentration, typically 140 μg/mL. A series of two-folddilutions were then prepared from the 140 μg/mL solution and the diluteddrug was added to the cells at 1:1 (v/v) according to the diagram shownin FIG. 11.

The plate is covered with a black lid and placed in the incubator forfour hours to allow absorption of the drug by the cells. The plate isthen removed from the incubator, the drug is aspirated, the wells arewashed with Dulbecco's phosphate buffered saline (DPBS) and fresh,pre-warmed medium is added to the wells. The columns of wells not to beirradiated are covered with foil strips, the black lid is exchanged fora clear lid, and the plate is then moved to the irradiation templatebeneath the light beam and irradiated for ten minutes, for a total of3.3 joules. The plate is returned to the incubator and assessed forviability using XTT after 24 hours. The wells covered with foil stripsare important controls for the activity of Photofrin® in the absence ofirradiation and the effect of light on the cells in the absence of drug.

The optical absorbance of the wells in column 1 (high drugconcentration, no cells) is evaluated for absorption at 450 nm to ensurethat the drug is not interfering with the XTT assay. The percentviability of each of the wells in columns 2-9 and 11 is calculatedrelative to the mean value of column 10 (cell control, no drug, nolight). For each dose response obtained, a four parameter logistic (4PL)nonlinear regression analysis is performed of the percent viabilityversus the log-Photofrin® concentration. The point of inflection of theregression curve (C value) is defined as the EC₅₀ and used in thecalculation of relative potency. Curve fitting and calculation of theEC₅₀ is performed with Softmax v5.4.2 (Molecular Devices, Inc.) or Prismv.5.04 (GraphPad, Software Inc.) software. The mean percent viability ofcolumns 9 and 11 is also calculated.

A typical assay result is shown in FIG. 12. The sigmoidal curve on theleft is the dose-response curve of a standard preparation of Photofrin®,while the curve on the right is the dose-response curve of a Photofrin®preparation containing half of the nominal activity. The ration of thestandard to test sample EC₅₀ is 0.5.

The invention described herein can be modified for the irradiation ofcells at different wavelengths by selection of the appropriate filters,including infrared or ultraviolet wavelengths. Depending on the celltype, drug, and filter efficiency, the drug absorption and irradiationtimes can be varied to obtain the desired results.

2. Detailed Methods Method for Thawing A549 Cells 1. Materials andReagents

-   -   1.1. A549 Human Lung Carcinoma (ATCC—Cat. No.: CCL-185)    -   1.2. 25 cm² Flasks (Corning—Cat. No.: 431463, or equivalent)    -   1.3. 75 cm² Flasks (Corning—Cat. No.: 431464, or equivalent)    -   1.4. 50 mL tubes (Fisher Scientific—Cat. No. 17-512F, or        equivalent)    -   1.5. Hemacytometer    -   1.6. RPMI-1640 (+) phenol red (Lonza—Cat. No.: 12-167F, or        equivalent)    -   1.7. HI-FBS (Gibco—Life Technologies—Cat No.: 10082-147, or        equivalent)    -   1.8. 200 mM L-Glutamine (Lonza—Cat. No.: 17-605E, or equivalent)    -   1.9. 0.25% Trypsin-EDTA (Gibco—Cat. No.: 25200-056, or        equivalent)

2. Instruments

-   -   2.1. Forma Scientific centrifuge with swinging bucket rotor        (Model 5682, or equivalent)    -   2.2. Water bath set at 37° C.    -   2.3. Humidified incubator set at 37° C. with 5% CO2

3. Prepared Reagents

-   -   3.1. RPMI-1640 Complete Media    -   3.1.1. 500 mL bottle of RPMI-1640 basal media containing phenol        red    -   3.1.2. Add 50 mL HI-FBS    -   3.1.3. Add 5.5 mL of 200 mM L-Glutamine

4. Procedure

-   -   4.1. Remove one vial of A549 lung carcinoma cells from liquid        nitrogen storage and after ensuring that the vial is tightly        closed, rapidly thaw vial by holding all but the top of the vial        submerged in a 37° C. water bath until the vial contents begins        to thaw but while ice still remains.    -   4.2. Liberally spray the vial with 70% ethanol, wipe with        tissue, and place into a laminar flow hood.    -   4.3. Transfer the contents of the vial to a 50 mL conical tube        containing 9 mL RPMI-1640 complete medium containing phenol red        (Step 3.1) (hereafter referred to as complete medium in this        method).    -   4.4. Centrifuge the tube in a swing bucket rotor set at 1200 RPM        (220 RCF) for five minutes at room temperature.    -   4.5. Discard supernatant and gently flick the tube to resuspend        the cells in the residual volume left in the tube.    -   4.6. Add 5 mL complete medium, transfer the cell solution to a        25 cm² flask, and then place the flask into a 37° C. incubator        with 5% CO2.    -   4.7. Monitor the flask daily and when the cells reach        approximately 80% confluence, remove the media, and wash the        cells by adding 2 mL DPBS to the flask. Ensure that all cells        are rinsed and discard the DPBS.    -   4.8. Add 1 mL 0.25% trypsin-EDTA to prewash the cells, tip flask        to coat all cells, and discard the trypsin.    -   4.9. Add another 1 mL trypsin, tip to coat all cells, and then        place into a 37° C. incubator for 4 minutes or until cells        detach.    -   4.10. Stop the trypsin reaction by adding 4 mL complete media to        the flask and then collect all cells and transfer to a 50 mL        conical tube.    -   4.11. Count the cell solution using a hemacytometer and seed the        cells into the appropriate number of 75 cm² flasks as needed        using the following seeding densities:

Day Cell Needed Seeding Density For Day 3 Add 0.4 × 10E+06 cells/75 cm2flask For Day 4 Add 0.3 × 10E+06 cells/75 cm2 flask For Day 5 Add 0.25 ×1E+06 cells/75 cm2 flask

-   -   4.12. When the cells in the 75 cm² are ready to seed into 96        well assay plates (approximately 80% confluent), remove the        media from the flask.    -   4.13. Add 5 mLs DPBS to the flask, swirl to rinse all cells and        then remove and discard the DPBS.    -   4.14. Add 2 mLs 0.25% Trypsin-EDTA to the flask, tip to coat all        cells and then remove and discard the trypsin.    -   4.15. Add another 2 mLs 0.25% Trypsin-EDTA and again, tip to        coat all cells and then place the flask into the 37° C.        incubator for 6 minutes or until cells detach.    -   4.16. Once the cells are detached, add 8 mLs of complete media,        collect the cells and transfer the volume to a 50 mL conical        tube.    -   4.17. Proceed to “Method for Seeding A549 Cells into 96 Well        Assay Plates.”

Method for Passaging A549 Cells 1. Materials and Reagents

-   -   1.1. 1549 Human Lung Carcinoma (ATCC—Cat. No.: CCL—185)    -   1.2. 25 cm² Flasks (Corning—Cat. No. 431463, or equivalent)    -   1.3. 75 cm² Flasks (Corning—Cat. No. 431464, or equivalent)    -   1.4. 50 mL tubes (Fisher Scientific—Cat. No.: 14-432-22, or        equivalent)    -   1.5. DPBS (Lonza—Cat. No.: 17-512F, or equivalent)    -   1.6. Hemacytometer    -   1.7. RPMI-1640 (+) phenol red (Lonza—Cat. No.: 12-167F, or        equivalent)    -   1.8. HI-FBS (Gibco—Life Technologies—Cat. No.: 10082-147, or        equivalent)    -   1.9. 200 mM L-Glutamine (Lonza—Cat. No.: 17-605E, or equivalent)    -   1.10. 0.25% Trypsin-EDTA (Gibco—Cat. No.: 25200-056, or        equivalent)

2. Instruments

-   -   2.1. Forma Scientific centrifuge with winging bucket rotor        (Model 5682, or equivalent)    -   2.2. Water bath set at 37° C.    -   2.3. Humidified incubator set at 37° C. with 5% Co2

3. Prepared Reagents

-   -   3.1. RPMI-1640 Complete Media    -   3.2. 500 mL bottle of RPMI-1640 basal media containing phenol        red    -   3.3. Add 50 mL HI-FBS    -   3.4. Add 5.5 mL of 200 mM L-Glutamine

4. Procedure

-   -   4.1. Monitor the flask daily and when the cells reach        approximately 80% confluence, remove the media and wash the        cells by adding 2 mL DPBS to the flask. Ensure that all cells        are rinsed and discard the DPBS.    -   4.2. Add 1 mL 0.25% trypsin-EDTA to prewash the cells, tip flask        to coat all cells and discard the trypsin.    -   4.3. Add another 1 mL trypsin, tip to coat all cells and then        place into a 37° C. incubator for 4 minutes or until cells        detach.    -   4.4. Stop the trypsin reaction by adding 4 mL complete media to        the flask and then collect all cells and transfer to a 50 mL        conical tube.    -   4.5. Count the cell solution using a heacytometer and seed the        cells into the appropriate number of 75 cm² flasks as needed        using the following seeding densities:

Day Cell Needed Seeding Density For Day 3 Add 0.4 × 10E+06 cells/75 cm2flask For Day 4 Add 0.3 × 10E+06 cells/75 cm2 flask For Day 5 Add 0.25 ×1E+06 cells/75 cm2 flask

-   -   4.6. When the cells in the 75 cm² are ready to seed into 96 well        assay plates (approximately 80% confluent), remove the media        from the flask.    -   4.7. Add 5 mLs DPBS to the flask, swirl to risen all cells and        then remove and discard the DPBS.    -   4.8. Add 2 mLs 0.25% Trypsin-EDTA to the flask, tip to coat all        cells and then remove and discard the trypsin.    -   4.9. Add another 2 mLs 0.25% Trypsin-EDTA and again, tip to coat        all cells and then place the flask into the 37° C. incubator for        6 minutes or until cells detach.    -   4.10. Once the cells are detached, add 8 mLs of complete media,        collect the cells and transfer the volume to a 50 mL conical        tube.    -   4.11. Proceed to “Method for Seeding A549 Cells Into 96 Well        Assay Plates.”        Method for Seeding A549 Cells into 96 Well Assay Plates

1. Materials and Reagents

-   -   1.1. 75 cm² Flasks (Corning—Cat. No.: 431464, or equivalent)    -   1.2. 50 mL tubes (Fisher Scientific—Cat. No.: 14-432-22, or        equivalent)    -   1.3. DPBS (Lonza—Cat. No.: 17-512F, or equivalent)    -   1.4. Hemacytometer (Fisher Scientific—Cat. No.: S17040, or        equivalent)    -   1.5. RPMI-1640 (+) phenol red (Lonza—Cat. No.: 12-167F, or        equivalent)    -   1.6. RPMI-1640 (−) phenol red (Lonza—Cat. No.: 12-918F, or        equivalent)    -   1.7. Heat-Inactivated FBS (Gibco—Life Technologies—Cat. No.:        10082-147, or equivalent)    -   1.8. 200 mM L-Glutamine (Lonza—Cat. No.: 17-605E, or equivalent)    -   1.9. 0.25% Trypsin-EDTA (Gibco—Cat. No.: 25200-056, or        equivalent)    -   1.10. Black 96 well tissue culture-treated assay plates with        universal black lids (Costar 3916 and Corning 3935        respectively)*    -   1.11. 50 mL reagent reservoir (Fisher Scientific—Cat. No.:        07-200-127, or equivalent)

2. Instruments

-   -   2.1. Forma Scientific centrifuge with swinging bucket rotor        (Model: 5682, or equivalent)    -   2.2. Water bath set at 37° C. with 5% CO2    -   2.3. Finnpipette 12 channel digital pipettor (Fisher        Scientific—Cat. No.: 21-377-830, or equivalent)

3. Prepared Reagents

-   -   3.1. RPMI-1640 Complete Media with or without phenol red        -   3.1.1. 500 mL bottle of RPMI-1640 basal media        -   3.1.2. Add 50 mL heat-inactivated fetal bovine serum        -   3.1.3. Add 5.5 mL of 200 mM L-Glutamine

4. Procedure

-   -   4.1. Cells should be approximately 80% confluent in a 75 cm²        flask.    -   4.2. Remove the medium and wash the cells by adding 5 mL DPBS to        the flask. Ensure that all cells are rinsed and then remove and        discard the DPBS.    -   4.3. Add 2 mL 0.25% trypsin-EDTA to prewash the cells, tip flask        to coat all cells and then remove and discard the trypsin.    -   4.4. Add another 2 mL trypsin, tip to coat all cells and then        place into a 37° C. incubator for 6 minutes or until cells        detach.    -   4.5. Stop the trypsin reaction by adding 8 mL complete medium to        the flask and then collect all cells and transfer to a 50 mL        conical tube.    -   4.6. Count the cell solution using a hemacytometer and record        the cell density.        -   4.6.1. Cell density=______ cells/mL    -   4.7. Calculate the number of cells needed for the assay based on        the number of plates.        -   4.7.1. ______ plates×1E+06 cells/plate=______ cells needed    -   4.8. Calculate the volume of cell suspension required and        transfer to 50 mL conical tube.        -   4.8.1. Cells needed (Step 4.7)/______ cell density (Step            4.6)=______ mL of cell suspension to transfer    -   4.9. Add 10-20 mL of complete medium and centrifuge the tubes at        220×g for 5 minutes.    -   4.10. Discard the supernatant and then sharply flick the pellet        to resuspend the cells in the residual volume left in the tube.    -   4.11. Resuspend the cells in the appropriate volume of complete        media based on the number of assay plates:        -   4.11.1. ______ plates×10 mL/plate=______ mL complete medium    -   4.12. Transfer the suspended cell volume to a 50 mL reservoir        and use a multi-channel pipettor to add 100 μL of the cell        suspension to the appropriate wells in the 96 well plates        according to the following template.        NOTE: Ensure cells are evenly dispersed during this step,        resuspending as necessary with pipettor.

1 2 3 4 5 6 7 8 9 10 11 12 DPBS DPBS DPBS DPBS DPBS DPBS DPBS DPBS DPBSDPBS DPBS DPBS DC Cells Cells Cells Cells Cells Cells Cells Cells CellsCells MC DC Cells Cells Cells Cells Cells Cells Cells Cells Cells CellsMC DC Cells Cells Cells Cells Cells Cells Cells Cells Cells Cells MC DCCells Cells Cells Cells Cells Cells Cells Cells Cells Cells MC DC CellsCells Cells Cells Cells Cells Cells Cells Cells Cells MC DC Cells CellsCells Cells Cells Cells Cells Cells Cells Cells MC DPBS DPBS DPBS DPBSDPBS DPBS DPBS DPBS DPBS DPBS DPBS DPBS

-   -   4.13. Add 200 μL of DPBS to all wells of the top and bottom rows        (rows A and H in the plates).    -   4.14. Add 200 μL of complete media without phenol red to the        media control wells (MC).    -   4.15. Add 100 μL of complete media without phenol red to the        drug control wells (DC).    -   4.16. Switch the clear covers that came with the black plates        for black covers and place plates into a humidified incubator        set at 37° C. with 5% CO for 24 hours.        NOTE: Retain the clear cover in a sterile environment for the        irradiation step in “Method for Irradiating A549 Cells in 96        Well Assay Plates.”        Method for Treating A549 Cells with Photofrin®

1. Materials and Reagents

-   -   1.1. 15 mL tubes (Fisher Scientific—Cat. No.: 14-959-49B, or        equivalent)    -   1.2. RPMI-1640 basal media without phenol red (Lonza—Cat. No.:        12-918F, or equivalent)    -   1.3. 50 mL reagent reservoir (Fisher Scientific—Cat. No.:        07-200-127, or equivalent)    -   1.4. 0.9% Sterile saline solution (Teknova—S5819, or equivalent)    -   1.5. 96 well polypropylene dilution plates (Fischer        Scientific—Cat. No.: 07-200-745)    -   1.6. Photofrin® vials

2. Instruments

-   -   2.1. Water bath set at 37° C.    -   2.2. Humidified incubator set at 37° C. with 5% CO2    -   2.3. Finnpipette 12 channel digital pipettor (Fischer        Scientific—Cat. No.: 21-377-830, or equivalent)    -   2.4. Benchtop rotating shaker (Hoefer Red Rotor, or equivalent)

3. Prepared Reagents

-   -   3.1. RPMI-1640 Complete Medium without phenol red (Complete        Medium)        -   3.1.1. 500 mL bottle of RPMI-1640 basal medium        -   3.1.2. Add 50 m Heat-Inactivated FBS        -   3.1.3. Add 5.5 mL of 200 mM L-Glutamine

4. Procedure

-   -   4.1. Place RPMI-1640 complete media without phenol red (complete        medium) into a water bath set at 37° C. for 1 hour prior to        beginning treatment.    -   4.2. All steps are performed aseptically in a biosafety cabinet.    -   4.3. Label four 50 mL conical tubes as follows: Working        Reference, Working Test and Cell Control, and Test Concentrate.    -   4.4. Aliquot 31.8 mL sterile saline for injection into the        Photofrin® tube and set aside.    -   4.5. Calculate the volume of complete medium needed based on the        number of assay plates and add the calculated volume to each of        the labeled 50 mL conical tubes.        -   4.5.1. ______ plates×4.72 mL=______ mL per tube    -   4.6. Calculate the volume of saline for injection or Photofrin®        solution to be diluted based on the number of assay plates and        add the calculated volume to tube labeled Cell Control and mix        well.        -   4.6.1. ______ plates×280 μL=______ μL    -   4.7. Use the following template to set-up the assay dilution        plate.        -   4.7.1.1.1. [insert table]    -   4.8. Add approximately 15 mL of complete medium to a reservoir.    -   4.9. Using a multichannel pipettor, dispense 125 μL of complete        media to wells B3-G8 of a dilution plate. Use a separate        dilution plate for each assay plate.    -   4.10. Dispose of the remaining medium in the reservoir and        transfer the volume from the Cell Control tube to the reservoir        and then add 125 μL to wells B9-G10 of the dilution plate.    -   4.11. Darken the working environment using window coverings as        necessary to achieve ambient light less than 0.3 mW/cm² to        ensure that the Photofrin® is not unintentionally activated.    -   4.12. Remove sufficient aliquots of the Reference Standard from        the freezer and thaw in a 37° C. water bath, disinfect with 70%        isopropanol and place in the biosafety cabinet.    -   4.13. Remove a vial of Photofrin® form the box, remove the metal        seal from the top of the vial, tap the vial on the hood surface        to clear powder from the rubber cap and then carefully remove        the rubber cap and place inverted on the surface of the hood.    -   4.14. Using a 10 mL pipet, transfer the contents of the conical        tube labeled T directly to the Photofrin® vial, replace the        rubber cap and while holding the vial at the top and bottom,        invert multiple times to mix well.    -   4.15. Pour the volume back into the 50 mL tube labeled Test        Concentrate.    -   4.16. Add the appropriate volume calculated in Step 4.6 from the        tube labeled T to the tube labeled Working Test and mix well.    -   4.17. Mix the contents well and add the appropriate volume        calculated in Step 4.6 from the aliquot of Reference Standard to        the tube labeled Working Reference and mix well.    -   4.18. Prepare a 50 mL reservoir and then transfer the contents        from the Working Reference tube to the reservoir.    -   4.19. Using a multi-channel pipettor, dispense 125 μL to wells        B1-D2 of the dilution plate.    -   4.20. Add an additional 125 μL to wells B2-D2 such that the        total volume added for those wells is 250 μL.    -   4.21. Add 125 μL to wells B11-D11 in the dilution plate.    -   4.22. Repeat for each of the assay plates and then discard the        remaining volume and reservoir.    -   4.23. Prepare another 50 mL reservoir and then transfer the        contents from the Working Test tube to the reservoir.    -   4.24. Using a multi-channel pipettor, dispense 125 μL to wells        E1-G2 of the dilution plate.    -   4.25. Add an additional 125 μL to wells E2-G2 such that the        total volume added for those wells is 250 μL.    -   4.26. Repeat for each of the assay plates and then discard the        remaining volume and reservoir.    -   4.27. Using a multi-channel pipettor with 6 channels/tips, mix        the well volumes in wells B2-G2 of the dilution plate three        times and then transfer 125 μL of this volume to wells B3-G3 to        create the first dilution.    -   4.28. Repeat this dilution scheme with wells B8-G8. After        transferring and mixing the volume in wells B8-G8, discard 125        μL instead of transferring to B9-G9.    -   4.29. Take the plate with the seeded A549 cells from the        incubator and place into biosafety cabinet. Ensure that the cell        plate and dilution plates are correctly aligned and then use the        multi-channel pipettor fitted with 11 tips to transfer 100 μL        from wells A1-D11 of the dilution plate to the corresponding row        of the cell plate. Before pulling the volume up, mix each row        gently 3 times in the dilution plate and then transfer the        volume to the cell plate.    -   4.30. Use the multi-channel pipettor fitted with 11 tips to        transfer 100 μL from wells E1-H11 of the dilution plate to the        corresponding row of the cell plate. Before pulling the volume        up, mix each row gently 3 times in the dilution plate and then        transfer the volume to the cell plate.    -   4.31. Replace black cover on the cell plates and place onto a        bench-top rotary shaker with the rotation set on low and start        the shaker for a total of 20 rotations to mix the well volumes.    -   4.32. Return the plates to the incubator for 4 hours and then        proceed with “Method for Irradiating Photofrin® —treated A549        Cells in 96 Well Plates.”

Method for Irradiation Photofrin® —Treated A549 Cells in 96 WellPlates 1. Materials and Reagents

-   -   1.1. DPBS (Lonza—catalog #: 17-512F, or equivalent)    -   1.2. RPMI-1640 (−) phenol red (Lonza—catalog #: 12-918F, or        equivalent)    -   1.3. Retained clear lids from black 96 well tissue        culture-treated assay plates (Costar 3916)    -   1.4. 50 mL reagent reservoir (Fisher Scientific—catalog #:        07-200-127, or equivalent)    -   1.5. Laser safety goggles    -   1.6. Foil adhesive plate seals cut into ¾ inch strips        (Thermo-Fisher—catalog #: AB-0626)    -   1.7. Ice bucket with half water and half ice    -   1.8. Waste container for discarded well volumes

2. Instruments

-   -   2.1. Water bath set at 37° C.    -   2.2. Humidified incubator set at 37° C. with 5% CO2    -   2.3. Finnpipette 12 channel digital pipettor (Fisher        Scientific—catalog #: 21-377-830, or equivalent)    -   2.4. 1000 Watt Arc Lamp Power Supply (Oriel Model 68820)    -   2.5. 1000 Watt Xenon ozone-free arc lamp in a lamp housing with        2 inch condenser (Newport 6271 and Oriel 66921 respectively)    -   2.6. Newport 2 inch aluminum liquid filter (Newport 6123)    -   2.7. Mirror holder, beam turning assembly—2 inch series        (Newport 66246) with 400-630 nm dichroic mirror (Newport 66229)    -   2.8. Lenses (Newport KPX229 76.2 diameter×200 mm focal length)    -   2.9. Multiple filter holder, 2 In Series, 2×0.75 in Max Thick,        1.8 in Clear Aperture (Newport 6215) fitted with 50% infrared        blocking filter (Newport 59042) and 650 nm cut-off filter        (Andover Corporation Part No. 650-FL07-50)    -   2.10. Multiple Filter Holder, 3 In Series, 3×0.75 in Max Thick,        2.8 in Clear Aperture (Newport 6216)    -   2.11. Plastic washer spacers (Newport 6215-503)    -   2.12. Peristaltic pump fitted with ⅛″ i.d. Tygon tubing for        cooling aluminum liquid filter    -   2.13. Timer

3. Prepared Reagents

-   -   3.1. RPMI-1640 Complete Media without phenol red        -   3.1.1. 500 mL bottle of RPMI-1640 basal medium        -   3.1.2. Add 50 mL HI-FBS        -   3.1.3. Add 5.5 mL of 200 mM L-Glutamine

4. Procedure

-   -   4.1. Place RPMI-1640 complete medium without phenol red        (hereafter referred to as complete medium in this method) and        DPBS into a water bath set at 37° C. for 1 hour prior to        beginning irradiation procedures.    -   4.2. At least 45 minutes prior to irradiation, perform the        following steps:        -   4.2.1. Dispense about ⅓ volume water into an ice bucket and            then add a half volume of ice.        -   4.2.2. Place the flow tubing into the ice bucket and            submerge the tubing and then fill the ice bucket to the top            with ice.        -   4.2.3. Turn on the circulator and start the circulating            water to at least a 6 mL/minute flow rate.    -   4.3. 15 minutes prior to irradiation, perform the following        steps:        -   4.3.1. Turn on the Xenon lamp power supply and press the            “Output Pre-Adjust” button and ensure that the needle moves            to 1000 Watts.        -   4.3.2. Put on eye protection and then press the “Lamp Start”            button on the power supply to ignite the lamp.        -   4.3.3. Prepare adhesive foil strips for covering the control            wells.    -   4.4. After the cells have been incubated with Photofrin® for 4        hours, darken the room and then take the cell plates from the        incubator and place into a biosafety cabinet.    -   4.5. Using the pre-warmed solutions, fill one reservoir with the        DPBS and one with the complete medium.    -   4.6. Using a multi-channel pipettor fitted with 12 tips, remove        and discard the contents of wells A1-D12, taking care to keep        the tips at the edge of the well to minimize monolayer        disruption. Discard the tips.    -   4.7. Using a multi-channel pipettor fitted with 12 tips, remove        and discard the contents of wells E1-H12, taking care to keep        the tips at the edge of the well to minimize monolayer        disruption. Discard the tips.    -   4.8. Carefully add back 200 μL of the pre-warded DPBS to the        wells to wash out the drug and then remove and discard this        volume.    -   4.9. Slowly add back 200 μL of the pre-warmed complete medium to        all of the wells.    -   4.10. Cover the wells A10-H11 using the adhesive foil strips,        taking care that the entire column is blocked and that adjacent        non-control columns are not.    -   4.11. Replace the black plate cover with a clear one and move        the plate to the template stage under the lamp and start        irradiation for 10 minutes.    -   4.12. After 10 minutes, return the plate to the hood, remove the        foil adhesive and replace the black lid.    -   4.13. Return the plates to the incubator for 24 hours and then        proceed with “Method for XTT staining of Photofrin®-treated A549        cells.”

Method for XTT Staining of Photofrin® —Treated A549 Cells in 96 WellAssay Plates 1. Materials and Reagents

-   -   1.1. 50 mL tubes (Fisher Scientific—Cat. No. 14-432-22, or        equivalent)    -   1.2. DPBS (Lonza—Cat. No.: 17-512F, or equivalent)    -   1.3. 50 mL reagent reservoir (Fisher Scientific—Cat. No.:        07-200-127, or equivalent)    -   1.4. XTT sodium salt (Sigma—Cat No.: X4626, or equivalent)    -   1.5. Phenazine methosulfate (Sigma—Cat. No.: P9625)

2. Instruments

-   -   2.1. Water bath set at 37° C.    -   2.2. Humidified incubator set at 37° C. with 5% CO2    -   2.3. Finnpipette 12 channel digital pipettor (Fisher        Scientific—Cat. No.: 21-377-830, or equivalent)    -   2.4. Spectramax 340PC plate reader

3. Prepared Reagents

-   -   3.1. XTT staining solution        -   3.1.1. Calculate the appropriate amount of XTT powder            needed.            -   3.1.1.1. ______ plates×5 mg/plate=______ total mg XTT                needed            -   3.1.1.2. Total volume of DPBS needed=mg XTT=______ mL        -   3.1.2. Dissolve the XTT powder in the required volume of            pre-warmed DPBS.        -   3.1.3. Add 40 μL of stock PMS to each mL of dissolved XTT            powder.            -   3.1.3.1. ______ mL XTT solution×40 μL of stock                PMS=______ μL of stock PMS

4. Procedure

-   -   4.1. Remove the 96 well treated plates from the incubator and        place into the hood.    -   4.2. Dispense the XTT staining solution into a 50 mL reservoir.    -   4.3. Add 50 μL of the XTT staining solution directly to the        wells of the plate and then place the plate into the incubator        for 4 or more hours until color change has developed fully.    -   4.4. After color development, check the wells for bubbles and        remove these using a flamer or other method.    -   4.5. Read the plates on a Spectramax plate reader and record the        OD values using a 430 nm wavelength.

3. Results of Studies Conducted to Determine the Feasibility of an InVitro Potency Assay

An optical train is constructed allowing the even illumination of a96-well tissue culture plate. Treatment of cells with Photofrin®followed by irradiation at 400-650 nm results in Photofrin®dose-dependent killing of the cells. An assay system for determining thepotency of a test lot of Photofrin® relative to a Reference Standard isestablished and evaluated for linearity, specificity, and robustness.

Materials and Methods Cells and Media

A549 human lung carcinoma cells are obtained from ATCC and grown in RPMImedium with 10% heat-inactivated fetal bovine serum and 2% L-glutamine.

Reagents and Materials

Photofrin® (Lot No.: 0G067, expiration date Oct. 31, 2012) is obtainedfrom Pinnacle Biologics, Inc. SN-38 is from LKT Labs (Cat. No. CO154;St. Paul, Minn.). Carboplatin is from Sigma (Cat. No. C2538), andPaclitaxel is from Sigma (Cat. No. T1912).

Summary of Procedure

The irradiation apparatus used is shown in FIGS. 1-9 and describedabove. An overview of the test method is provided in Example 2. Adetailed description of the test method is provided in Example 3.

Results Irradiation Apparatus

The Xenon arc lamp produces a bright, broad wavelength irradiance beamthat when collimated still retains a bright center. This lack ofhomogeneity in the intensity of the light beam has to be minimized inorder to achieve even irradiation over the area of the 96-well plate.For this purpose, two plano-convex lenses are introduced into the lightpath to approximate a Kohler illumination (FIG. 1). After an initialdosing ranging study of Photofrin® (data not shown), each well of anassay plate containing A549 cells is treated equally with Photofrin® at1.5 μg/mL to assess homogeneity of the irradiation. For this experiment,the percent viability is calculated relative to a second plate of cellswithout drug. The degree of cell killing is observed to varysignificantly with position on the plate, with wells in the middle ofthe plate showing no killing (103% viability). To address theinhomogeneity in irradiance, the distance from the final bandpass filterto the sample is raised approximately 2-fold from 54 cm to 95.5 cm whilethe first surface of the plano-convex lenses is maintained at 13.6 cmfrom the 650 nm cut-off filter. By increasing the length of the lightpath, the center zone of irradiance is broadened to cover the area ofthe entire plate (FIG. 13). As the light path is lengthened, the overallirradiance is decreased, necessitating the use of a higher Photofrin®concentration.

Effect of Plate Type

Initial experiments are performed using standard clear 96-well plates.When black plates with black lids are used to perform the assay, ahigher EC₅₀ is obtained, indicating that extraneous light is reachingthe cells in the clear plates and causing additional cell killing (seeFIG. 14A and FIG. 14B, Table 1). Use of black plates results in aconsistent 4-fold increase in the EC₅₀ relative to clear plates.

TABLE 1 Effect of plate type on Photofrin ® EC₅₀ Experiment Clear PlateEC₅₀ Black plate EC₅₀ PIN-004 1.87 8.08 PIN-008 2.49 10.1 2.46 9.48 2.339.59 Mean 2.29 9.31 Data from two experiments is shown. The doseresponse curves of Photofrin ® were either nine point (250-0.977 μg/m,PIN-004) or six point (30.0-1.09 μg/mL, PIN-008).

Effect of Chemotherapeutic Drugs

The broadly active chemotherapeutics drugs SN-38 (20 μM top dose), theactive metabolite of irinotecan, carboplatin (7.5 mM top dose), andpaclitaxel (20 μM top dose) are tested to determine if sufficient cellkilling can be achieved under the anticipated assay conditions, i.e., 24hour incubation with XTT endpoint. None of the drugs produce sufficientcell killing under the assay conditions to allow their use a positive orsystem suitability control (data not shown).

Selection of Assay Endpoint

XTT and MTT are two types of formazan dyes that are used to measuremitochondrial function as a surrogate for cell viability. A Photofrin®dose response curve is performed with either XTT or MTT as the endpoint.The results obtained with these two endpoints differ by approximately15%, but each produces consistent values (see Table 2, below). As thepotency of an unknown will be determined with respect to a ReferenceLot, the absolute EC₅₀ value is not critical. Given that the use of theMTT dye requires an additional solubilization step, XTT is selected foruse in this assay.

TABLE 2 Comparison of XTT and MTT assay endpoints Experiment EC₅₀ - XTTEC₅₀ - MTT PIN-009 9.48 8.28 9.42 8.11 Mean 9.45 8.20

Positional Effects

The effect of sample position within the 96-well is investigated. Theexperimental design includes a series of plates where the position oftwo samples and the controls is altered. The two samples represent a“Standard” sample where the high test concentration on the plate is 70μg/mL, and a sub-potent “Test” sample where the standard is diluted afurther two-fold with medium so that the high test concentration is 35μg/mL. When the regression analysis is performed, both samples aretreated as if the high test concentration is 70 μg/mL, thereby makingthe Test sample appear to have a potency of one-half of the Standard.The sample arrangement for the various plates is described in Table 3below. The results of the analysis are compiled in Table 4 below.

TABLE 3 Sample arrangement for positional effects Sample PlacementPlates 1, 4 Standard, 70 μg/mL, columns 2-8, left to right dilutionTest, 35 μg/mL, columns 2-8, left to right dilution Controls in columns9-11 Plate 2 Test, 35 μg/mL, columns 2-8, left to right dilutionStandard, 70 μg/mL, columns 2-8, left to right dilution Controls incolumns 9-11 Plate 3 Standard, 70 μg/mL, columns 2-8, right to leftdilution Test, 35 μg/mL, columns 2-8, right to left dilution Controls incolumns 9-11 Plate 5 Standard, 70 μg/mL, left to right dilution Test, 35μg/mL, left to right dilution Controls in columns 2-4

TABLE 4 Results of positional analysis Standard Test Sample EC₅₀ SampleEC₅₀ −D, +L/CC +D, −L/CC Plate 1 11.1 21.2 1.00 0.994 Plate 2 10.7 22.20.941 0.926 Plate 3 11.2 21.5 0.992 0.981 Plate 4 11.0 21.5 0.947 0.928Plate 5 10.8 20.1 0.985 1.00 Mean 11.0 21.3 0.973 0.967 CV 1.89 3.592.78 3.74 Mean Potency 0.516 Each plate was assayed identically afterset-up as described in Table 3. The EC₅₀ for each of the two samples oneach plate was calculated using Prism. The mean EC₅₀ and the coefficientof variation (CV) for each sample across all of the plates werecalculated. The mean potency was calculated as the ratio of the EC₅₀(standard)/EC₅₀ (test). The ratio of the no drug, irradiated (−D, +L)and with 70 μg/mL drug, no irradiation (+D, −L) to the cell control (CC,no drug, no irradiation) were also calculated.

Assay Linearity

Assay linearity is tested by comparing the experimentally derivedpotencies to the theoretical potencies of a series of super- andsub-potent test samples. To best mimic test samples of varyingpotencies, Photofrin® is dissolved in one-fourth the nominal dissolutionvolume to create a sample with a theoretical potency of 4. Furtherdilution of this sample in saline is performed to create samples withnominal potencies of 2, 1, 0.5, and 0.25. Each of the five samples isthen handled identically as they are diluted with medium prior toaddition to the assay plates for potency determination relative to anominally prepared sample representing a reference lot. The result ofthese analyses is shown in Table 5, below. At the extreme of thepotencies tested, 4 and 0.25, excessive or insufficient cell killingoccurs and the EC₅₀ can either not be determine or is determined with awide confidence interval.

These results indicate that lots of test sample with apparent potenciesof greater than two or less than 0.5 should be reviewed carefully, andadjustments in the preparation of the test sample should be consideredto obtain an appropriate amount of cell killing under the assayconditions.

TABLE 5 Assay linearity Ratio of Theoretical EC₅₀ ExperimentalTheoretical/Experimental Potency STD EC₅₀ Test Potency Potency 4 10.32.36* 4.36* 0.917* 2 10.6 5.16 2.05 0.976 0.5 11.2, 21.3, 21.7 0.526,0.516 0.951, 0.969 11.2 0.25 11.6 NC* NC NC Theoretical and experimentalpotencies for Photofrin ®. Theoretical potency reflects the predictedpotency based on the sample preparation. EC₅₀ values for the standardsample (70 μg/mL high test drug concentration) and Test (variable hightest drug concentration) are shown with the experimentally derivedpotency obtained as the ratio of the EC₅₀(standard)/EC₅₀(test). *Theconfidence interval on the EC₅₀ is wide due to poor curve fitting. **Notcalculable due to poor curve fitting.

Robustness Testing

In the following series of experiments, key assay parameters aredeliberately varied to determine the effect of small variations on assayperformance. The variations are designed to encompass the normalvariation that could be encountered during the routine performance ofthe assay.

Cell Seeding Density

The number of cells seeded into each well of the 96-well plate is variedfrom the nominal value of 1.0×10⁴ cells/well. The densities tested are0.8×10⁴, 1.0×10⁴, and 1.2×10⁴ cells/well. A Standard sample (70 μg/mLhigh test concentration) and a Test sample (35 μg/mL high testconcentration) is assayed on each plate. The expected potency of theTest sample is 0.5. The results of this experiment are shown in Table 6below. The results indicated that the assay may be sensitive to cellseeding density, and that densities about 1.0×10⁴ may lead to inaccuratepotency determination.

TABLE 6 Effect of assay plate seeding density Seeding Density EC₅₀ STDEC₅₀ Test Potency of Test Sample* 0.8 × 10⁴ 11.0 21.1 0.521 cells/well1.9 × 10⁴ 11.2 21.3 0.526 cells/well 1.2 × 10⁴ 10.9 27.9 0.391cells/well EC₅₀ values for the standard sample (70 μg/mL high test drugconcentration) and Test (35 μg/mL high test drug concentration) areshown with the experimentally derived potency obtained as the ratio ofthe EC₅₀(standard)/EC₅₀(test). *The expected potency for each of theconditions was 0.5.

Cell Passage Number

When A549 cells are removed from liquid nitrogen storage, the initialpassage number is designated at P1. These cells are continuouslypassaged during the execution of the experiments described in thisreport. In preparation for this aspect of robustness testing, a secondvial of cells is thawed and cultured. This experiment compares theresults obtained with A549 cells at Passage 10 (P10) with those atpassage 17 (P17). A Standard sample (70 μg/mL high test concentration)and a Test sample (35 μg/mL high test concentration) is assayed on eachplate. The expected potency of the Test sample is 0.5. The results ofthis experiment are shown in Table 7 below. The P10 data for the Testsample fails to result in a well-fit regression resulting in a wideconfidence interval for the EC₅₀ value, nevertheless, the calculatedpotency value is close to the expected value of 0.5. Importantly, theEC₅₀ values and the calculated potency of the assay performed with theP17 cells is consistent with P10 data in the assay, and the overallperformance of the assay, demonstrating the suitability of the P17cells.

TABLE 7 Effect of A549 cell passage number Seeding Passage EC₅₀ STD EC₅₀Test Potency of Test Sample* P10 10.5 18.9** 0.556 P17 10.1 19.2 0.526EC₅₀ values for the Standard sample (70 μg/mL high test drugconcentration) and Test (35 μg/mL high test drug concentration) areshown with the experimentally derived potency calculated as the ratio ofthe EC₅₀(standard)/EC₅₀(test). *The expected potency for each of theconditions was 0.5. **Wide confidence interval due to poor curvefitting.

Time of Irradiation

The time of irradiation is varied from the nominal value of 10 minutesto include assays performed at 8, 9, 10, 11, and 12 minutes of lightirradiation. A Standard sample (70 μg/mL high test concentration) and aTest sample (35 μg/mL high test concentration) is assayed on each plate.The expected potency of the Test sample is 0.5. The results of thisexperiment are shown in Table 8 below. The results indicate a trendtoward lower EC₅₀ values with increasing irradiation time. The effectappears proportional in both samples and potencies within 11% of theexpected value of 0.5 is obtained under all of the conditions.

TABLE 8 Effect irradiation time Irradiation Time EC₅₀ STD EC₅₀ TestPotency of Test Sample*  8 minutes 12.0 24.5 0.490  9 minutes 12.0 25.20.476 10 minutes 11.1 22.8 0.487 11 minutes 11.6 20.9 0.555 12 minutes10.8 21.0 0.514 EC₅₀ values for the Standard sample (70 μg/mL high testdrug concentration) and Test (35 μg/mL high test drug concentration) areshown with the experimentally derived potency calculated as the ratio ofthe EC₅₀(standard)/EC₅₀(test). *The expected potency for each of theconditions was 0.5.

Time of Photofrin® Absorption

The amount of time that Photofrin® is allowed to be in contact with thecells is varied from the nominal value of 4 hours to include assaysperformed at 3.5, 4, and 4.5 hours of Photofrin® absorption. A Standardsample (70 μg/mL high test concentration) and a Test sample (35 μg/mLhigh test concentration) is assayed on each plate. The expected potencyof the Test sample is 0.5. The results of this experiment are shown inTable 9 below. Comparison of the EC₅₀ values and the calculatedpotencies indicate that incubation with Photofrin® for 4.5 hours mayaffect the absolute value of the EC₅₀, but the potency value obtainedwas within 15% of the expected value of 0.5.

TABLE 9 Effect Photofrin ® absorption time Absorption Time EC₅₀ STD EC₅₀Test Potency of Test Sample* 3.5 hours 10.4 19.5 0.533 4.0 hours 10.220.1 0.507 4.5 hours 9.23 16.1 0.573 EC₅₀ values for the Standard sample(70 μg/mL high test drug concentration) and Test (35 μg/mL high testdrug concentration) are shown with the experimentally derived potencycalculated as the ratio of the EC₅₀(standard)/EC₅₀(test). *The expectedpotency for each of the conditions was 0.5.

Post-Irradiation Incubation Time

The amount of time that irradiated plates are incubated beforeassessment of cell killing is varied from the nominal value of 24 hoursto include assays performed with 22, 24, and 26 hours of incubationpost-irradiation. A Standard sample (70 μg/mL high test concentration)and a Test sample (35 μg/mL high test concentration) is assayed on eachplate. The expected potency of the Test sample is 0.5. The results ofthis experiment are shown in Table 10 below. Comparison of the EC₅₀values and the calculated potencies indicate that post-irradiationincubation times from 22-26 hours produce potencies within 15% of theexpected value.

TABLE 10 Effect post-irradiation incubation time Post-irradiation TimeEC₅₀ STD EC₅₀ Test Potency of Test Sample* 22 hours 9.16 16.7 0.549 24hours 10.2 18.9 0.54 26 hours 9.55 16.6 0.575 EC₅₀ values for theStandard sample (70 μg/mL high test drug concentration) and Test (35μg/mL high test drug concentration) are shown with the experimentallyderived potency calculated as the ratio of theEC₅₀(standard)/EC₅₀(test). *The expected potency for each of theconditions was 0.5.

Post-Irradiation Incubation Temperature

The temperature that irradiated plates are incubated before assessmentof cell killing is varied from the nominal value of 37° C. to includeassays performed at 35° C., 37° C., and 39° C. A Standard sample (70μg/mL high test concentration) and a Test sample (35 μg/mL high testconcentration) is assayed on each plate. The expected potency of theTest sample is 0.5. The study is conducted in two experiments, each witha nominal temperature assay and one test temperature assay. The resultsof this experiment are shown in Table 11 below. The results indicatethat post-irradiation temperatures between 35° C. and 39° C. have littleeffect on the EC₅₀ or calculated potency of the samples.

TABLE 11 Effect post-irradiation incubation temperature Post-irradiationPotency of Test temperature EC₅₀ STD EC₅₀ Test Sample* 35° C. 9.68 21.60.448 37° C. 10.8 19.9 0.543 37° C. 9.9 20.0 0.495 39° C. 8.32 16.10.516 EC₅₀ values for the Standard sample (70 μg/mL high test drugconcentration) and Test (35 μg/mL high test drug concentration) areshown with the experimentally derived potency calculated as the ratio ofthe EC₅₀(standard)/EC₅₀(test). *The expected potency for each of theconditions was 0.5.

Different Operators

The assay feasibility is performed primarily by KP, who can beconsidered an expert in performing the assay. For this study, a secondoperator, AB, is trained by watching KP perform and assay and then byperforming an assay using two assay plates (AB Pilot-1, and -2). AB thenperforms an assay in parallel with KP to assess inter-operatorvariability. Each operator assays in parallel two plates containing aStandard sample (70 μg/mL high test concentration) and a Test sample (35μg/mL high test concentration). The expected potency of the Test sampleis 0.5. The results of this experiment are shown in Table 12 below.There are inter-operator differences in the absolute value of the EC₅₀obtained for each sample, with AB consistently obtaining lower valuesthan KP. No clear bias is observed, however, in the calculated potenciesfor the samples with all of the potency values within 13% of theexpected value of 0.5. This suggests that the assay EC₅₀ values may besensitive to inter-operator variability, but that the calculated potencyvalue is robust. As the EC₅₀, for a Reference Lot to be included in thesystem suitability criteria, inter-operator differences in the EC₅₀values should be minimized through a root cause investigation withappropriate corrective action.

TABLE 12 Inter-operator variability EC₅₀ STD EC₅₀ Test Potency of TestSample* Operator AB Pilot Assay-1 6.62 16.1 0.411 AB Pilot Assay-2 6.7612.6 0.537 Inter-operator Assay AB-1 6.37 13.7 0.465 AB-2 8.23 14.60.564 Mean 7.30 14.2 0.515 KP-1 9.45** 20.1 0.470 KP-1 9.96 19.8 0.503Mean 0.971 20.0 04.87 EC₅₀ values for the Standard sample (70 μg/mL hightest drug concentration) and Test (35 μg/mL high test drugconcentration) are shown with the experimentally derived potencycalculated as the ratio of the EC₅₀(standard)/EC₅₀(test). *The expectedpotency for each of the conditions was 0.5. **Wide confidence intervaldue to poor curve fitting.

Summary: Behavior of the Control Samples

Critical aspects of the system suitability will include the acceptableperformance of the assay controls. The expectation is that the viabilityof cells receiving no drug but with light (−D, +L) and cells receiving ahigh concentration of drug but no light (+D, −L) should approximate theviability of the cell control not receiving drug or light (CC). Theresults of this analysis for the experiments described in this report,where applicable, are shown in Table 13 below. The percent viability ofall samples is based on the CC, which is calculated set at 1.00 and isnot included in the Table. The data shows that both of these controlsare well-behaved with a mean value equal to the CC, and a coefficient ofvariation of 6-7%.

TABLE 13 Behavior of controls Experiment −D, +L +D, −L Table 1 1.11 1.050.931 0.893 1.11 1.09 1.20

1.10

1.08

Table 2 1.11 1.06 0.994 0.947 1.06 1.06 0.997 0.986 Table 4 1.00 0.9940.941 0.926 0.992 0.981 0.947 0.928 0.985 1.00 Table 5 0.977 0.999 0.9601.02 0.941 1.00 0.904 1.29 0.901 0.973 Table 6 0.993 0.990 0.994 0.9810.993 0.977 Table 7 0.998 0.973 0.996 1.00 Table 8 0.991 1.02 0.9820.992 0.986 0.995 0.942 0.959 0.952 0.985 Table 9 1.02 1.01 1.03 0.8981.06 0.959 Table 10 1.00 1.09 1.00 1.08 0.989 0.995 Table 11 0.994 0.9800.994 0.973 1.04 1.02 1.03 0.956 Table 12 0.906 0.990 0.937 0.906 1.001.03 1.08 1.02 1.02 1.00 1.01 1.02 Mean 1.0 1.00 CV 0.060 0.064 Percentviability of the indicated controls is tabulated. Shaded values for +D,−L originated from clear plates and showed significant cell killing andwere excluded from the analysis. Black plates and lids were used inobtaining all other data in this table.

1. A lighting system comprising: a lamp housing including a lamp and alight-port, wherein broad spectrum light from the lamp exits the lamphousing through the light-port; a first lens to collimate the broadspectrum light that exits the lamp housing through the light-port; aninfrared absorbing filter to pass a first portion of the collimatedbroad spectrum light and absorb infrared light of the broad spectrumlight that passes through the light-port and that is collimated by thefirst lens, wherein the first portion of the collimated broad spectrumlight comprises a second portion of the collimated broad spectrum light;an optical filter to pass the second portion of the collimated broadspectrum light after the first portion of the collimated broad spectrumlight reaches the optical filter; and a second lens to disperse thesecond portion of the collimated light that passed through the opticalfilter.
 2. (canceled)
 3. (canceled)
 4. (canceled)
 5. (canceled) 6.(canceled)
 7. (canceled)
 8. (canceled)
 9. (canceled)
 10. (canceled) 11.(canceled)
 12. (canceled)
 13. (canceled)
 14. (canceled)
 15. (canceled)16. (canceled)
 17. (canceled)
 18. (canceled)
 19. (canceled) 20.(canceled)
 21. (canceled)
 22. (canceled)
 23. (canceled)
 24. (canceled)25. (canceled)
 26. (canceled)
 27. (canceled)
 28. A method comprising:using the lighting system of claim 1 to irradiate contents of a cellculture plate.
 29. (canceled)
 30. A method for studying aphotosensitizer, the method comprising: adding the photosensitizer to aportion of wells on a cell culture plate to form photosensitizer assaywells, the wells comprising carcinoma cells; incubating thephotosensitizer assay wells for a first predetermined time period;optionally washing the photosensitizer assay wells; irradiating thephotosensitizer assay wells with the lighting system of claim 1 at apredetermined wavelength to form irradiated wells, wherein each well isuniformly irradiated; incubating the irradiated wells for a secondpredetermined time period; and determining percent viability of thecarcinoma cells contained in the wells.
 31. (canceled)
 32. (canceled)33. (canceled)
 34. (canceled)
 35. (canceled)
 36. (canceled) 37.(canceled)
 38. (canceled)
 39. (canceled)
 40. The lighting system ofclaim 1, wherein the first lens comprises a condenser lens or a Fresnellens.
 41. The lighting system of claim 1, wherein the optical filtercomprises an infrared blocking filter and a short pass filter.
 42. Thelighting system of claim 41, wherein the infrared blocking filterabsorbs residual infrared light of the first portion of the collimatedbroad spectrum light, and wherein the short pass filter filters outlight of the first portion of the collimated broad spectrum light ofwavelengths greater than 650 nm.
 43. The lighting system of claim 41,further comprising: a reflector to reflect the first portion of thecollimated broad spectrum light that passes through the infraredabsorbing filter.
 44. The lighting system of claim 43, wherein the firstportion of the collimated broad spectrum light reflected by thereflector propagates to the infrared blocking filter, and wherein thesecond portion of the collimated broad spectrum light that propagates tothe infrared blocking filter, as part of the first portion of thecollimated broad spectrum light, passes through the infrared blockingfilter and then through the short pass filter.
 45. The lighting systemof claim 43, wherein the first portion of the collimated broad spectrumlight reflected by the reflector propagates to the short pass filter,and wherein the second portion of the collimated broad spectrum lightthat propagates to the short pass filter, as part of the first portionof the collimated broad spectrum light, passes through the short passfilter and then through the infrared blocking filter.
 46. The lightingsystem of claim 43, wherein the reflector comprises a dichroic mirror,and wherein the dichroic mirror absorbs at least a portion of infraredlight that passes through the light-port and the infrared absorbingfilter.
 47. The lighting system of claim 1, wherein the lamp housing issealed so that less than 1% of the broad spectrum light from the lampexits the lamp housing other than through the light-port.
 48. Thelighting system of claim 1, further comprising: a ring stand; a firstsupport ring removably attached to the ring stand; and a base.
 49. Thelighting system of claim 48, wherein the second lens comprises a firstdispersing lens and a second dispersing lens; and wherein the firstsupport ring holds the first dispersing lens and the second dispersinglens in place.
 50. The lighting system of claim 49, wherein the firstdispersing lens comprises a first plano convex lens; wherein the seconddispersing lens comprises a second plano convex lens; wherein the firstplano convex lens comprises a first plano side and a first convex side;wherein the second plano convex lens comprises a second plano side and asecond convex side; and wherein the first convex side is adjacent to thesecond convex side with a gap between the first convex side and thesecond convex side.
 51. The lighting system of claim 1, furthercomprising: a wall, wherein the light-port is located within the wall,and wherein a position of the second lens is adjustable in at least oneof a direction parallel to the wall and a direction perpendicular to thewall.
 52. The lighting system of claim 51, further comprising: a shelf,and a lens slider including a first hole for passing light; wherein theshelf comprises a shelf riser parallel to the wall, wherein the shelfcomprises a shelf top perpendicular to the wall, wherein the shelf topincludes a second hole for passing light, wherein the second lens isremovably attached to the lens slider, wherein the lens slider isremovably attached to the shelf top, and wherein at least a portion ofthe first hole is above or below at least a portion of the second hole.53. The lighting system of claim 1, wherein the lamp housing comprises atop, wherein the light-port is within the top, wherein the first lens iswithin the light-port, and wherein the infrared absorbing filter, theoptical filter, and the second lens are located at positions above thetop.
 54. The lighting system of claim 1, wherein the lamp housingcomprises a bottom, wherein the light-port is within the bottom, whereinthe first lens is within the light-port, and wherein the infraredabsorbing filter, the optical filter, and the second lens are located atpositions below the bottom.
 55. The method according to claim 30,wherein the photosensitizer is a porphyrin-based anti-neoplastic agent.56. The method according to claim 30, wherein the photosensitizer assaywells are irradiated with light within a range of 400 nm to 650 nm.